Methods and reagents for isolating biologically active peptides

ABSTRACT

One aspect of the present invention is the synthesis of a binary method that combines variegated peptide display libraries, e.g., in a “display mode”, with soluble secreted peptide libraries, e.g., in a “secretion mode”, to yield a method for the efficient isolation of peptides having a desired biological activity.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/174,943, filed on Oct. 19, 1998, the specification of which isincorporated herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] High throughput screening has become a dominant tool in thepharmaceutical industry for the discovery of lead compounds that can bemodified into candidates for drug development. For instance, it isabundantly used for identification of ligands with high affinity forreceptors. In this regard, combinatorial techniques have providedapproaches to generating and deconvoluting large libraries of testcompounds in high throughput screens. It involves selection andamplification of a subset of molecules with desired biologicalproperties from complex libraries.

[0003] One technique which has emerged for identification of peptideleads involves the use of peptide display methodologies such as phagedisplay. Phage-displayed peptide libraries can comprise vast collectionsof short, randomized polypeptides that are displayed on the surface of afilamentous bacteriophage particle. Thus, each “peptide” is actually theN-terminal sequence of a phage-coat protein, that is encoded by arandomly-mutated region of the phage genome responsible for theproduction of the coat protein. In this manner, each unique peptide inthe library is physically linked with the DNA molecule encoding it.Antibodies and other binding molecules can be used as “targets” tospecifically select rare phage clones bearing ligand peptides, andsequencing of the corresponding viral DNA will reveal their amino acidsequences. Relatively high-affinity peptides for a variety of peptide-and non-peptide-binding targets have been affinity-isolated from epitopelibraries. This technology has been used to map epitopes on proteins andto find peptide mimics for a variety of target molecules. Many powerfulapplications can be envisioned in the areas of drug design and thedevelopment of diagnostic markers, vaccines and toleragens. For thepurposes of drug discovery, there are potential advantages in the use ofgenetically encoded libraries, such as phage display (Scott et al,Science 249, 386 (1990); Devlin et al., Science 249, 386 (1990)),“peptide on plasmid” (Cull et al. PNAS 89, 1865 (1992)), and in vitrotranslation-based systems (Mattheakis et al. PNAS 91, 9022 (1994)),compared to the use of synthetic small molecule libraries (Bunin et al.PNAS 91, 4708 (1994); Gordon et al. J. Med. Chem. 37, 1385 (1994); andDooley et al., Science 266, 2019 (1994)). The genetic encoding oflibraries allows the resynthesis and rescreening of molecules with adesired binding activity. The resulting amplification of interactingmolecules in subsequent rounds of selection can lead to the isolation ofextremely rare, specific binders from a large pool of molecules.

[0004] However, despite the success of these methods, they suffer fromnumerous sources of error and bias, such as very low initialconcentrations of species, non-specific binding, and, significantly, thesampling of only a fraction of the library at the end of an experiment.

SUMMARY OF THE INVENTION

[0005] One aspect of the invention provides a method for generating apeptide with a selected biological activity, comprising the steps of:

[0006] (i) providing a peptide display library comprising a variegatedpopulation of test peptides expressed on the surface of a population ofdisplay packages;

[0007] (ii) in a display mode, isolating, from the peptide displaylibary, a sub-population of display packages enriched for test peptideswhich have a desired binding specificity and/or affinity for a cell or acomponent thereof;

[0008] (iii) in a secretion mode, simultaneously expressing the enrichedtest peptide sub-population under conditions wherein the test peptidesare secreted and are free of the display packages; and

[0009] (iv) assessing the ability of the secreted test peptides toregulate a biological process in a target cell.

[0010] For instance, the peptide display library can be a phage displaylibrary, e.g., which utilizes phage particles such as M13, f1, fd, If1,Ike, Xf, Pf1, Pf3, X, T4, T7, P2, P4, φX-174, MS2 or f2. In preferredembodiments, the phage display library is generated with a filamentousbacteriophage specific for Escherichia coli and the phage coat proteinis coat protein III or coat protein VIII. For instance, the filamentousbacteriophage can be M13, fd, and f1.

[0011] In other embodiments, the peptide display library is a bacterialcell-surface display library or a spore display library.

[0012] In certain embodiments, the test peptides are enriched from thepeptide display library in the display mode by a differential bindingmeans comprising affinity separation of test peptides which specificallybind the cell or component thereof from test peptides which do not. Forexample, the differential binding means can include panning the peptidedisplay library on whole cells, affinity chromatographic means in whicha component of a cell is provided as part of an insoluble matrix (.e.g,a cell surface protein attached to a polymeric support), and/orimmunoprecipitating the display packages.

[0013] In the display mode, the test peptides can be enriched for thosewhich bind to a cell-type specific marker and/or a cell surface receptorprotein. For example, the test peptide library can be enriched in thedisplay mode for test peptides which bind to a G-protein coupledreceptor, such as a chemoattractant peptide receptor, a neuropeptidereceptor, a light receptor, a neurotransmitter receptor, a cyclic AMPreceptor, or a polypeptide hormone receptor. In other embodiments, thetest peptide library can be enriched in the display mode for testpeptides which bind to a receptor tyrosine kinase, such as an EPHreceptor. In still other embodiments, the test peptide library can beenriched in the display mode for test peptides which bind to a cytokinereceptor or an MIRR receptor. In certain embodiments, the test peptidelibrary can be enriched in the display mode for test peptides which bindto an orphan receptor.

[0014] In preferred embodiments, the peptide display library includes atleast 10³ different test peptides.

[0015] In preferred embodiments, the test peptides are 4-20 amino acidresidues in length.

[0016] In certain embodiments, each of the test peptides are encoded bya chimeric gene comprising (i) a coding sequence for the test peptide,(ii) a coding sequence for a surface protein of the display package fordisplaying the test peptides on the surface of a population of displaypackages, and (iii) RNA splice sites flanking the coding sequence forthe surface protein, wherein, in the display mode, the chimeric gene isexpressed as fusion protein including the test peptide and the surfaceprotein, whereas in the secretion mode, the test peptide is expressedwithout the surface protein as a result of the coding sequence for thesurface protein being removed by RNA splicing.

[0017] In preferred embodiments, the test peptides are expressed by aeukaryotic cell, more preferably a mammalian cell, in the secretionmode.

[0018] In preferred embodiments, the target cell is a eukaryotic cell,more preferably a mammalian cell such as a human cell.

[0019] In certain embodiments, the biological process scored for in thesecretion mode includes a change in cell proliferation, celldifferentiation or cell death. In other embodiments, the biologicalprocess which is detected is changes in intracellular calciummobilization, intracellular protein phosphorylation, phospholipidmetabolism, and/or expression of cell-specific marker genes.

[0020] In certain embodiments, the target cell includes a reporter geneconstruct containing a reporter gene in operative linkage with one ormore transcriptional regulatory elements responsive to the signaltransduction acitivity of the cell surface receptor protein, expressionof the reporter gene providing the detectable signal. For instance, thereporter gene can encode a gene product that gives rise to a detectablesignal selected from the group consisting of: color, fluorescence,luminescence, cell viability relief of a cell nutritional requirement,cell growth, and drug resistance. In preferred embodiments, the reportergene encodes a gene product selected from the group consisting ofchloramphenicol acetyl transferase, beta-galactosidase and secretedalkaline phosphatase. In other preferred embodiments, the reporter geneencodes a gene product which confers a growth signal.

[0021] In certain embodiments, the secretion mode includes assessing theability of the secreted test peptides to inhibit the biological activityof an exogenously added compound on the target cells.

[0022] In an exemplary embodiment: in step (ii) above, display packageswhich bind to endothelial cells are isolated; and in step (iv) above,the ability of the secreted test peptides to inhibit proliferation ofendothelial cells is assessed. For example, in step (iv) the ability ofthe secreted test peptides to inhibit proliferation of endothelial cellsin the presense of an angiogenic amount of an endogenous growth factorcan be assessed.

[0023] The subject invention also specifically contemplates thatpeptides identified in the secretion mode can be converted intopeptidomimietics.

[0024] Moreover, in certain embodiments, the subject method includes thefurther step of formulating, with a pharmaceutically acceptable carrier,one or more test peptides which regulate the biological process in thetarget cell or peptidomimetics thereof.

[0025] Another aspect of the present invention provides a peptidedisplay library enriched for test peptides having a desired bindingspecificity and/or affinity for a cell or a component thereof and whichregulate a biological process in a target cell.

[0026] Still another aspect of the present invention relates to a vectorcomprising a chimeric gene for a chimeric protein, which chimeric genecomprises (i) a coding sequence for a test peptide, (ii) a codingsequence for a surface protein of a display package, and (iii) RNAsplice sites flanking the coding sequence for the surface protein,wherein,

[0027] in a display mode, the chimeric gene is expressed as a fusionprotein including the test peptide and the surface protein such that thetest peptide can be displayed on the surface of a population of displaypackages,

[0028] whereas in the secretion mode, the test peptide is expressedwithout the surface protein as a result of the coding sequence for thesurface protein being removed by RNA splicing.

[0029] In certain embodiments, the chimeric gene can include a secretionsignal sequence for secretion of the test peptide in the secretion mode,e.g., secretion of the test peptide from eukaryotic cells, preferablymammalian cells.

[0030] Yet another aspect of the present invention provides a vectorlibrary, each vector comprising a chimeric gene for a chimeric protein,which chimeric gene comprises (i) a coding sequence for a test peptide,(ii) a coding sequence for a surface protein of a display package, and(iii) RNA splice sites flanking the coding sequence for the surfaceprotein, wherein,

[0031] in a display mode, the chimeric gene is expressed as fusionprotein including the test peptide and the surface protein such that thetest peptide can be displayed on the surface of a population of displaypackages,

[0032] whereas in the secretion mode, the test peptide is expressedwithout the surface protein as a result of the coding sequence for thesurface protein being removed by RNA splicing,

[0033] the vector library collectively encodes a variegated populationof test peptides.

[0034] In preferred embodiments, the vector library collectively encodesat least 10³ different test peptides.

[0035] In preferred embodiments, the test peptides are 4-20 amino acidresidues in length.

[0036] Another aspect of the present invention is a cell compositioncomprising a population of cells containing the vector library describedabove.

[0037] Still another aspect of the present invention provides a methodfor generating a peptide with a selected antimicrobial activity,comprising the steps of:

[0038] (i) providing a recombinant host cell population which expressesa soluble peptide library comprising a variegated population of testpeptides;

[0039] (ii) culturing the host cells with a target microorganism underconditions wherein the peptide library is secreted and diffuses to thetarget microorganism; and

[0040] (iii) selected host cells expressing test peptides that inhibitgrowth of the target microorganism.

[0041] For example, the target microorganism is a bacteria or a fungus.In certain embodiments, the host cells are cultured on agar embeddedwith the, target microorganisms. For example, antimicrobial activity ofa test peptide can be determined by zone clearing in the agar.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1: Schematic of pAM6 M13/COS peptide expression plasmid.

[0043]FIG. 2: Schematic of pAM7 & pAM9 M13/COS peptide expressionplasmid.

[0044]FIG. 3: Schematic of pAM8 M13/COS peptide expression plasmid.

[0045]FIG. 4A: Transwell cell culture chamber.

[0046]FIG. 4B: Indicator plate antimicrobial assay.

[0047]FIG. 5: Flowchart depicting utilization of the M13 display/COSsecretion method for identification of anti-angiogenic peptides.

[0048]FIG. 6: Nucleotide level depiction of pAM6 M13/COS peptideexpression plasmid.

[0049]FIG. 7: Nucleotide level depiction of pAM7 M13/COS peptideexpression plasmid.

[0050]FIG. 8: Nucleotide level depiction of pAM8 M13/COS peptideexpression plasmid.

[0051]FIG. 9: Expression of the Myc epit6pe-pVII in E. coli.

[0052]FIG. 10: Anti-myc western blot detection of myc-pVIII incorporatedinto phagemid capsids.

[0053]FIG. 11: Titration of plaque and colony forming units generatedupon phagemid rescue.

[0054]FIG. 12: Secretion of M13/COS plasmid encoded proteins from COS-7cells.

[0055]FIG. 13: Anti-pIll western blot detection of peptides incorporatedinto M13 phagemid capsids as pIII fusions

[0056]FIG. 14: Specific binding of pAM9-K1 phagemids to bovine capillaryendothelial cells

[0057]FIG. 15: Inhibition BCE cell proliferation in Transwells bypeptides secreted from COS-7 cells

DETAILED DESCRIPTION OF THE INVENTION

[0058] I. Overview

[0059] The present invention makes available a powerful directedapproach for isolating biologically active peptides. One aspect of thepresent invention is the synthesis of a binary method that combinesvariegated peptide display libraries, e.g., in a “display mode”, withsoluble secreted peptide libraries, e.g., in a “secretion mode”, toyield a method for the efficient isolation of peptides having a desiredbiological activity.

[0060] Utilizing peptide display techniques, a peptide library can firstbe reduced in complexity by panning or other affinity purificationtechniques. In particular, the subject method selects peptides having acertain affinity profile, e.g., a specificity and/or binding affinityfor a discrete cell or protein or other cellular component thereof by(i) displaying the peptides on the outer surface of a replicable geneticdisplay package to create a peptide display library, and (ii) usingaffinity selection techniques to enrich the population of displaypackages for those containing peptides which have a desired bindingspecificity for the target cell or cellular component (hereincollectively referred to as the “target”).

[0061] After the affinity enrichment step, the resulting sub-library isthen utilized in a secretion mode whereby the test peptides are secretedas soluble extracellular factors and their effect as paracrine orautocrine factors is scored. That is, the secretion mode measuresbiological activity of the test peptides in order to distinguish betweenagonist, antagonist, and inactive peptides with regard to regulating aparticular biological response of a test cell or tissue.

[0062] In preferred embodiments, the display mode and secretion mode canbe carried out without the need to sub-clone the test peptide codingsequence into another vector. To illustrate, FIGS. 1-3 show exemplaryvectors for sequential use in both the display and secretion modes. Inbacterial cells, the vectors produce a fusion protein consisting of asecretion signal sequence, the test peptide and the remaining C-terminalportion of the gene VIII protein. The resulting chimeric protein iscapable of being incorporated into an M13 phage particle. However, inmammalian cells (such as COS cells), the M13 coding sequences areremoved from the mature mRNA by virtue of splice sites which flank thephage sequence. Thus, the mature mRNA, in mammalian cells, encodes asecretion signal sequence and test peptide alone, which is secreted as asoluble peptide from the cell.

[0063] One advantage to such embodiments of the subject method is theability to reduce loss of peptide sequences from the sub-library byeliminating sub-cloning steps.

[0064] In an exemplary embodiment, the subject method can be used toidentify peptides with anti-anigiogenic activity, e.g., the ability toreversibly inhibit proliferation of endothelial cells. In this regard,the present invention makes available a method for identifyingendothelial inhibitors which can be used to inhibit angiogenesis relateddiseases and modulating angiogenic processes. As used herein, the term“angiogenesis” means the generation of new blood vessels into a tissueor organ. Under normal physiological conditions, humans or animalsundergo angiogenesis only in very specific restricted situations. Forexample, angiogenesis is normally observed in wound healing. fetal andembryonal development and formation of the corpus luteum, endometriumand placenta. The term “endothelium” means a thin layer of flatepithelial cells that lines serous cavities, lymph vessels, and bloodvessels. For instance, peptides isolated by the subject method may beidentified by their ability to bind to endothelial cells and overcomethe angiogenic activity of endogenous growth factors such as bFGF, invitro.

[0065] II. Definitions

[0066] Before further description of the invention, certain termsemployed in the specification, examples and appended claims are, forconvenience, collected here.

[0067] The term “peptide” refers to an oligomer in which the monomersare amino acids (usually alpha-amino acids) joined together throughamide bonds. Peptides are two or more amino acid monomers long, but moreoften are between 5 to 10 amino acid monomers long and can be evenlonger, i.e. up to 20 amino acids or more, although peptides longer than20 amino acids are more likely to be called “polypeptides.” The term“protein” is well known in the art and usually refers to a very largepolypeptide, or set of associated homologous or heterologouspolypeptides, that has some biological function. For purposes of thepresent invention the terms “peptide,” “polypeptide,” and “protein” arelargely interchangeable as all three types can be used to generate thedisplay library and so are collectively referred to as peptides.

[0068] The term “simultaneously expressing” refers to the expression ofa representative population of a peptide library, e.g., at least 50percent, more preferably 75, 80, 85, 90, 95 or 98 percent of all thedifferent peptide sequences of a library.

[0069] The term “random peptide library” refers to a set of random orsemi-random peptides, as well as sets of fusion proteins containingthose random peptides (as applicable).

[0070] The term “effective amount” refers to an amount sufficient toinduce a statistically significant result.

[0071] The term “ligand” refers to a molecule that is recognized by aparticular protein, e.g., a receptor. Any agent bound by or reactingwith a protein is called a “ligand,” so the term encompasses thesubstrate of an enzyme and the reactants of a catalyzed reaction. Theterm “ligand” does not imply any particular molecular size or otherstructural or compositional feature other than that the substance inquestion is capable of binding or otherwise interacting with a protein.A “ligand” may serve either as the natural ligand to which the proteinbinds or as a functional analogue that may act as an agonist orantagonist.

[0072] The language “replicable genetic display package” or “displaypackage” describes a biological particle which has genetic informationproviding the particle with the ability to replicate. The package candisplay a fusion protein including a peptide derived from the variegatedpeptide library. The test peptide portion of the fusion protein ispresented by the display package in a context which permits the peptideto bind to a target that is contacted with the display package. Thedisplay package will generally be derived from a system that allows thesampling of very large variegated peptide libraries. The display packagecan be, for example, derived from vegetative bacterial cells, bacterialspores, and bacterial viruses.

[0073] The language “differential binding means”, as well as “affinityselection” and “affinity enrichment”, refer to the separation of membersof the peptide display library based on the differing abilities ofpeptides on the surface of each of the display packages of the libraryto bind to the target. The differential binding of a target by testpeptides of the display can be used in the affinity separation of thosepeptides which specifically bind the target from those which do not. Forexample, the affinity selection protocol can also include a pre- orpost-enrichment step wherein display packages capable of binding“background targets”, e.g., as a negative selection, are removed fromthe library. Examples of affinity selection means include affinitychromatography, immunoprecipitation, fluorescence activated cellsorting, agglutination, and plaque lifts. As described below, theaffinity chromatography includes bio-panning techniques using eitherpurified, immobilized target proteins or the like, as well as wholecells.

[0074] The phrases “individually selective manner” and “individuallyselective binding”, with respect to binding of a test peptide with atarget protein, refers to the binding of a peptide to a certain proteintarget which binding is specific for, and dependent on, the molecularidentity of the protein target.

[0075] The term “solid support” refers to a material having a rigid orsemi-rigid surface. Such materials will preferably take the form ofsmall beads, pellets, disks, chips, dishes, multi-well plates, wafers orthe like, although other forms may be used. In some embodiments, atleast one surface of the substrate will be substantially flat. The term“surface” refers to any generally two-dimensional structure on a solidsubstrate and may have steps, ridges, kinks, terraces, and the likewithout ceasing to be a surface.

[0076] In an exemplary embodiment of the present invention, the displaypackage is a phage particle which comprises a peptide fusion coatprotein that includes the amino acid sequence of a test peptide. Thus, alibrary of replicable phage vectors, especially phagemids (as definedherein), encoding a library of peptide fusion coat proteins is generatedand used to transform suitable host cells. Phage particles formed fromthe chimeric protein can be separated by affinity selection based on theability of the peptide associated with a particular phage particle tospecifically bind a target. In a preferred embodiment, each individualphage particle of the library includes a copy of the correspondingphagemid encoding the peptide fusion coat protein displayed on thesurface of that package. Exemplary phage for generating the presentvariegated peptide libraries include M13, f1, fd, If1, Ike, Xf, Pf1,Pf3, X, T4, T7, P2, P4, φX-174, MS2 and f2.

[0077] The language “fusion protein” and “chimeric protein” areart-recognized terms which are used interchangeably herein, and includecontiguous polypeptides comprising a first polypeptide covalently linkedvia an amide bond to one or more amino acid sequences which definepolypeptide domains that are foreign to and not substantially homologouswith any domain of the first polypeptide. One portion of the fusionprotein comprises a test peptide, e.g., which can be random orsemi-random. A second polypeptide portion of the fusion protein istypically derived from an outer surface protein or display anchorprotein which directs the “display package” (as hereafter defined) toassociate the test peptide with its outer surface. As described below,where the display package is a phage, this anchor protein can be derivedfrom a surface protein native to the genetic package, such as a viralcoat protein. Where the fusion protein comprises a viral coat proteinand a test peptide, it will be referred to as a “peptide fusion coatprotein”. The fusion protein further comprises a signal sequence, whichis a short length of amino acid sequence at the amino terminal end ofthe fusion protein, that directs at least the portion of the fusionprotein including the test peptide to be secreted from the cytosol of acell and localized on the extracellular side of the cell membrane.

[0078] Gene constructs encoding fusion proteins are likewise referred toa “chimeric geries” or “fusion genes”.

[0079] The term “vector” refers to a DNA molecule, capable ofreplication in a host cell, into which a gene can be inserted toconstruct a recombinant DNA molecule.

[0080] The terms “phage vector” and “phagemid” are art-recognized andgenerally refer to a vector derived by modification of a phage genome,containing an origin of replication for a bacteriophage, and preferably,though optional, an origin (ori) for a bacterial plasmid. The use ofphage vectors rather than the phage genome itself provides greaterflexibility to vary the ratio of chimeric peptide/coat protein towild-type coat protein, as well as supplement the phage genes withadditional genes encoding other heterologous polypeptides, such as“auxiliary polypeptides” which may be useful in the “dual” peptidedisplay constructs described below.

[0081] The language “helper phage” describes a phage which is used toinfect cells containing a defective phage genome or phage vector andwhich functions to complement the defect. The defect can be one whichresults from removal or inactivation of phage genomic sequence requiredfor production of phage particles. Examples of helper phage are M13K07.

[0082] As used herein, “cell surface receptor” refers to molecules thatoccur on the surface of, cells, interact with the extracellularenvironment, and (directly or indirectly) transmit or transduce theinformation regarding the environment intracellularly in a manner thatmay modulate intracellular second messenger activities or transcriptionof specific promoters, resulting in transcription of specific genes.

[0083] As used herein, “extracellular signals” include a molecule orother change in the extracellular environment that is transducedintracellularly via cell surface proteins that interact, directly orindirectly, with the signal. An extracellular signal or effectormolecule includes any compound or substance that in some manner altersthe activity of a cell surface protein. Examples of such signalsinclude, but are not limited to, molecules such as acetylcholine, growthfactors and hormones, lipids, sugars and nucleotides that bind to cellsurface and/or intracellular receptors and ion channels and modulate theactivity of such receptors and channels.

[0084] As used herein, “extracellular signals” also include as yetunidentified substances that modulate the activity of a cellularreceptor, and thereby influence intracellular functions. Suchextracellular signals are potential pharmacological agents that may beused to treat specific diseases by modulating the activity of specificcell surface receptors.

[0085] “Orphan receptors” is a designation given to a receptors forwhich no specific natural ligand has been described and/or for which nofunction has been determined.

[0086] As used herein, a “reporter gene construct” is a nucleic acidthat includes a “reporter gene” operatively linked to at least onetranscriptional regulatory sequence. Transcription of the reporter geneis controlled by these sequences to which they are linked. The activityof at least one or more of these control sequences can be directly orindirectly regulated by the target receptor protein. Exemplarytranscriptional control sequences are promoter sequences. A reportergene is meant to include a promoter-reporter gene construct which isheterologously expressed in a cell.

[0087] The term “indicator gene” generically refers to an expressible(e.g., able to be transcribed and [optionally] translated) DNA sequencewhich is, for example, expressed in response to a signal transductionpathway modulated by a target receptor or ion channel. Exemplaryindicator genes include unmodified endogenous genes of the host cell,modified endogenous genes, or a reporter gene of a heterologousconstruct, e.g., as part of a reporter gene construct.

[0088] “Signal transduction” is the processing of physical or chemicalsignals from the cellular environment through the cell membrane, and mayoccur through one or more of several mechanisms, such asactivation/inactivation of enzymes (such as proteases, or other enzymeswhich may alter phosphorylation patterns or other post-translationalmodifications), activation of ion channels or intracellular ion stores,effector enzyme activation via guanine nucleotide binding proteinintermediates, formation of inositol phosphate, activation orinactivation of adenylyl cyclase, direct activation (or inhibition) of atranscriptional factor and/or activation.

[0089] The term “modulation of a signal transduction activity of areceptor protein” in its various grammatical forms, as used herein,designates induction and/or potentiation, as well as inhibition of oneor more signal transduction pathways downstream of a receptor.

[0090] Agonists and antagonists are “receptor effector” molecules thatmodulate signal transduction via a receptor. Receptor effector moleculesare capable of binding to the receptor, though not necessarily at thebinding site of the natural ligand. Receptor effectors can modulatesignal transduction when used alone, i.e. can be surrogate ligands, orcan alter signal transduction in the presence of the natural ligand,either to enhance or inhibit signaling by the natural ligand. Forexample, “antagonists” are molecules that block or decrease the signaltransduction activity of receptor, e.g., they can competitively,noncompetitively, and/or allosterically inhibit signal transduction fromthe receptor, whereas “agonists” potentiate, induce or otherwise enhancethe signal transduction activity of a receptor. The terms “receptoractivator” and “surrogate ligand” refer to an agonist which inducessignal transduction from a receptor.

[0091] The term “compound” as used herein is meant to include bothexogenously added test compounds and peptides expressed from a peptidelibrary.

[0092] III. Exemplary Embodiments

[0093] A. Display Mode

[0094] In its “display mode”, a library of test peptides is expressed bya population of display packages to form a peptide display library. Withrespect to the display package on which the variegated peptide libraryis manifest, it will be appreciated from the discussion provided hereinthat the display package will preferably be able to be (i) geneticallyaltered to encode heterologous peptide,. (ii) maintained and amplifiedin culture, (iii) manipulated to display the peptide-containing geneproduct in a manner permitting the peptide to interact with a targetduring an affinity separation step, and (iv) affinity separated whileretaining the nucleotide sequence encoding the test peptide (herein“peptide gene”) such that the sequence of the peptide gene can beobtained. In preferred embodiments, the display remains viable afteraffinity separation.

[0095] Ideally, the display package comprises a system that allows thesampling of very large variegated peptide display libraries, rapidsorting after each affinity separation round, and easy isolation of thepeptide gene from purified display packages or further manipulation ofthat sequence in the secretion mode. The most attractive candidates forthis type of screening are prokaryotic organisms and viruses, as theycan be amplified quickly, they are relatively easy to manipulate, andlarge number of clones can be created. Preferred display packagesinclude, for example, vegetative bacterial cells, bacterial spores, andmost preferably, bacterial viruses (especially DNA viruses). However,the present invention also contemplates the use of eukaryotic cells,including yeast and their spores, as potential display packages.

[0096] In addition to -commercially available kits for generating phagedisplay libraries (e.g. the Pharmacia Recombinant Phage Antibody System,catalog no. 27-9400-01; and the Stratagene SurZAP™ phage display kit,catalog no. 240612), examples of methods and reagents particularlyamenable for use in generating the variegated peptide display library ofthe present invention can be found in, for example, the Ladner et al.U.S. Pat. No. 5,223,409; the Kang et al. International Publication No.WO 92/18619; the Dower et al. International Publication No. WO 91/17271;the Winter et al. International Publication WO 92/20791; the Markland etal. International Publication No. WO 92/15679; the Breitling et al.International Publication WO 93/01288; the McCafferty et al.International Publication No. WO 92/01047; the Garrard et al.International Publication No. WO 92/09690; the Ladner et al.International Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al.(1991) PNAS 88:7978-7982. These systems can, with modificationsdescribed herein, be adapted for use in the subject method.

[0097] When the display is based on a bacterial cell, or a phage whichis assembled periplasmically, the display means of the package willcomprise at least two components. The first component is a secretionsignal which directs the recombinant peptide to be localized on theextracellular side of the cell membrane (of the host cell when thedisplay package is a phage). This secretion signal can be selected so asto be cleaved off by a signal peptidase to yield a processed, “mature”peptide. The second component is a display anchor protein which directsthe display package to associate the test peptide with its outersurface. As described below, this anchor protein can be derived from asurface or coat protein native to the genetic package.

[0098] When the display package is a bacterial spore, or a phage whoseprotein coating is assembled intracellularly, a secretion signaldirecting the peptide to the inner membrane of the host cell isunnecessary. In these cases, the means for arraying the variegatedpeptide library comprises a derivative of a spore or phage coat proteinamenable for use as a fusion protein.

[0099] In some instances it may be necessary to introduce anunstructured polypeptide linker region between portions of the chimericprotein, e.g., between the test peptide and display polypeptide. Thislinker can facilitate enhanced flexibility of the chimeric proteinallowing the test peptide to freely interact with a target by reducingsteric hindrance between the two fragments, as well as allowingappropriate folding of each portion to occur. The linker can be ofnatural origin, such as a sequence determined to exist in random coilbetween two domains of a protein. Alternatively, the linker can be ofsynthetic origin. For instance, the sequence (Gly4Ser)₃ can be used as asynthetic unstructured linker. Linkers of this type are described inHuston et al. (1988) PNAS 85:4879; and U.S. Pat. Nos. 5,091,513 and5,258,498. Naturally occurring unstructured linkers of human origin arepreferred as they reduce the risk of immunogenicity.

[0100] In the instance wherein the display package is a phage, thecloning site for the test peptide gene sequences in the phagemid shouldbe placed so that it does not substantially interfere with normal phagefunction. One such locus is the intergenic region as described by Zinderand Boeke, (1982) Gene 19: 1-10.

[0101] The number of possible combinations in a peptide library can getlarge as the length is increased and selection criteria for degeneratingat each position is relaxed. To sample as many combinations as possibledepends, in part, on the ability to recover large numbers oftransformants. For phage with plasmid-like forms (as filamentous phage),electrotransformation provides an efficiency comparable to that ofphage-transfection with in vitro packaging, in addition to a very highcapacity for DNA input. This allows large amounts of vector DNA to beused to obtain very large numbers of transformants. The method describedby Dower et al. (1988) Nucleic Acids Res., 16:6127-6145, for example,may be used to transform fd-tet derived recombinants at the rate ofabout 107 transformants/ug of ligated vector into E. coli (such asstrain MC1061), and libraries may be constructed in fd-tet B1 of up toabout 3×10⁸ members or more. Increasing DNA input and makingmodifications to the cloning protocol within the ability of the skilledartisan may produce increases of greater than about 10- fold in therecovery of transformants, providing libraries of up to 10¹⁰ or morerecombinants.

[0102] As will be apparent to those skilled in the art, in embodimentswherein high affinity peptides are sought, an important criteria for thepresent selection method can be that it is able to discriminate betweenpeptides of different affinity for a particular target, andpreferentially enrich for the peptides of highest affinity. Applying thewell known principles of peptide affinity and valence (i.e. avidity), itis understood that manipulating the display package to be renderedeffectively monovalent can allow affinity enrichment to be carried outfor generally higher binding affinities (i.e. binding constants in therange of 10⁶ to 10¹⁰ M⁻¹) as compared to the broader range of affinitiesisolable using a multivalent display package. To generate the monovalentdisplay, the natural (i.e. wild-type) form of the surface or coatprotein used to anchor the peptide to the display can be added at a highenough level that it almost entirely eliminates inclusion of the peptidefusion protein in the display package. Thus, a vast majority of thedisplay packages can be generated to include no more than one copy ofthe peptide fusion protein (see, for example, Garrad et al. (1991)Bio/Technology 9:1373-1377). In a preferred embodiment of a monovalentdisplay library, the library of display packages will comprise no morethan 5 to 10% polyvalent displays, and more preferably no more than 2%of the display will be polyvalent , and most preferably, no more than 1%polyvalent display packages in the population. The source of thewild-type anchor protein can be, for example, provided by a copy of thewild-type gene present on the same construct as the peptide fusionprotein, or provided by a separate construct altogether. However, itwill be equally clear that by similar manipulation, polyvalent displayscan be generated to isolate a broader range of binding affinities. Suchpeptides can be useful, for example, in purification protocols whereavidity can be desirable.

[0103] i) Phages as Display Packages

[0104] Bacteriophage are attractive prokaryotic-related organisms foruse in the subject method. Bacteriophage are excellent candidates forproviding a display system of the variegated peptide library as there islittle or no enzymatic activity associated with intact mature phage, andbecause their genes are inactive outside a bacterial host, rendering themature phage particles metabolically inert. In general, the phagesurface is a relatively simple structure. Phage can be grown easily inlarge numbers, they are amenable to the practical handling involved inmany potential mass screening programs, and they carry geneticinformation for their own synthesis within a small, simple package. Asthe peptide gene is inserted into the phage genome, choosing theappropriate phage to be employed in the subject method will generallydepend most on whether (i) the genome of the phage allows introductionof the peptide gene either by tolerating additional genetic material orby having replaceable genetic material; (ii) the virion is capable ofpackaging the genome after accepting the insertion or substitution ofgenetic material; and (iii) the display of the peptide on the phagesurface does not disrupt virion structure sufficiently to interfere withphage propagation.

[0105] One concern presented with the use of phage is that themorphogenetic pathway of the phage determines the environment in whichthe peptide will have opportunity to fold. Periplasmically assembledphage are preferred as the displayed peptides may contain essentialdisulfides, and such peptides may not fold correctly within a cell.However, in certain embodiments in which the display package formsintracellularly (e.g., where □ phage are used), it has been demonstratedin other instances that disulfide-containing peptides can assume properfolding after the phage is released from the cell.

[0106] Another concern related to the use of phage, but also pertinentto the use of bacterial cells and spores as well, is that multipleinfections could generate hybrid displays that carry the gene for oneparticular test peptide yet have two or more different test peptides ontheir surfaces. Therefore, it can be preferable, though optional, tominimize this possibility by infecting cells with phage under conditionsresulting in a low multiple-infection.

[0107] For a given bacteriophage, the preferred display means is aprotein that is present on the phage surface (e.g. a coat protein).Filamentous phage can be described by a helical lattice; isometricphage, by an icosahedral lattice. Each monomer of each major coatprotein sits on a lattice point and makes defined interactions with eachof its neighbors. Proteins that fit into the lattice by making some, butnot all, of the normal lattice contacts are likely to destabilize thevirion by aborting formation of the virion as well as by leaving gaps inthe virion so that the nucleic acid is not protected. Thus inbacteriophage, unlike the cases of bacteria and spores, it is generallyimportant to retain in the peptide fusion proteins those residues of thecoat protein that interact with other proteins in the virion. Forexample, when using the M13 cpVIII protein, the entire mature proteinwill generally be retained with the peptide fragment being added to theN-terminus of cpVIII, while on the other hand it can suffice to retainonly the last 100 carboxy terminal residues (or even fewer) of the M13cpIII coat protein in the peptide fusion protein.

[0108] Under the appropriate induction, the test peptide library isexpressed and exported, as part of the fusion protein, to the bacterialcytoplasm, such as when the □ phage is employed. The induction of thefusion protein(s) may be delayed until some replication of the phagegenome, synthesis of some of the phage structural-proteins, and assemblyof some phage particles has occurred. The assembled protein chains theninteract with the phage particles via the binding of the anchor proteinon the outer surface of the phage particle. The cells are lysed and thephage bearing the library-encoded test peptide (that corresponds to thespecific library sequences carried in the DNA of that phage) arereleased and isolated from the bacterial debris.

[0109] To enrich for and isolate phage which encodes a selected testpeptide, and thus to ultimately isolate the nucleic acid sequences (thepeptide gene) themselves, phage harvested from the bacterial debris areaffinity purified. As described below, when a test peptide whichspecifically binds a particular target is desired, the target can beused to retrieve phage displaying the desired test peptide. The phage soobtained may then be amplified by infecting into host cells. Additionalrounds of affinity enrichment followed by amplification may be employeduntil the desired level of enrichment is reached.

[0110] The enriched peptide-phage can also be screened with additionaldetection-techniques such as expression plaque (or colony) lift (see,e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeledtarget is used as a probe.

[0111] a) Filamentous Phage

[0112] Filamentous bacteriophages, which include M13, f1, fd, If1, Ike,Xf, Pf1, and Pf3, are a group of related viruses that infect bacteria.They are termed filamentous because they are long, thin particlescomprised of an elongated capsule that envelopes the deoxyribonucleicacid (DNA) that forms the bacteriophage genome. The F pIII filamentousbacteriophage (Ff phage) infect only gram-negative bacteria byspecifically adsorbing to the tip of F pIII, and include fd, fl and M13.

[0113] Compared to other bacteriophage, filamentous phage in general areattractive and M13 in particular is especially attractive because: (i)the 3-D structure of the virion is known; (ii) the processing of thecoat protein is well understood; (iii) the genome is expandable; (iv)the genome is small; (v) the sequence of the genome is known; (vi) thevirion is physically resistant to shear, heat, cold, urea, guanidiniumchloride, low pH, and high salt; (vii) the phage is a sequencing vectorso that sequencing is especially easy; (viii) antibiotic-resistancegenes have been cloned into the genome with predictable results (Hineset al. (1980) Gene 11:207-218); (ix) it is easily cultured and stored,with no unusual or expensive media requirements for the infected cells,(x) it has a high burst size, each infected cell yielding 100 to 1000M13 progeny after infection; and (xi) it is easily harvested andconcentrated (Salivar et al. (1964) Virology 24: 359-371). The entirelife cycle of the filamentous phage M13, a common cloning and sequencingvector, is well understood. The genetic structure of M13 is well known,including the complete sequence (Schaller et al. in The Single-StrandedDNA Phages eds. Denhardt et al. (NY: CSHL Press, 1978)), the identityand function of the ten genes, and the order of transcription andlocation of the promoters, as well as the physical structure of thevirion (Smith et al. (1985) Science 228:1315-1317; Raschad et al. (1986)Microbiol Dev 50:401-427; Kuhn et al. (1987) Science 238:1413-1415;Zimmerman et al. (1982) J Biol Chem 257:6529-6536; and Banner et al.(1981) Nature 289:814-816). Because the genome is small (6423 bp),cassette mutagenesis is practical on RF M13 (Current Protocols inMolecular Biology, eds. Ausubel et al. (NY: John Wiley & Sons, 1991)),as is single-stranded oligonucleotide directed mutagenesis (Fritz et al.in DNA Cloning, ed by Glover (Oxford, UK: IRC Press, 1985)). M13 is aplasmid and transformation system in itself, and an ideal sequencingvector. M13 can be grown on Rec-strains of E. coli. The M13 genome isexpandable (Messing et al. in The Single-Stranded DNA Phages, edsDenhardt et al. (NY: CSHL Press, 1978) pages 449-453; and Fritz et al.,supra) and M13 does not lyse cells. Extra genes can be inserted into M13and will be maintained in the viral genome in a stable manner.

[0114] The mature capsule or Ff phage is comprised of a coat of fivephage-encoded gene products: cpVIII, the major coat protein product ofgene VIII that forms the bulk of the capsule; and four minor coatproteins, cpIII and cpIV at one end of the capsule and cpVII and cpIX atthe other end of the capsule. The length of the capsule is formed by2500 to 3000 copies of cpVIII in an ordered helix array that forms thecharacteristic filament structure. The gene III-encoded protein (cpIII)is typically present in 4 to 6 copies at one end of the capsule andserves as the receptor for binding of the phage to its bacterial host inthe initial phase of infection. For detailed reviews of Ff phagestructure, see Rasched et al., Microbiol. Rev., 50:401-427 (1986); andModel et al., in The Bacteriophages, Volume 2, R. Calendar, Ed., PlenumPress, pp. 375-456 (1988).

[0115] The phage particle assembly involves extrusion of the viralgenome through the host cell's membrane. Prior to extrusion, the majorcoat protein cpVIII and the minor coat protein cpIII are synthesized andtransported to the host cell's membrane. Both cpVIII and cpIII areanchored in the host cell membrane prior to their incorporation into themature particle. In addition, the viral genome is produced and coatedwith cpV protein. During the extrusion process, cpV-coated genomic DNAis stripped of the cpV coat and simultaneously recoated with the maturecoat proteins.

[0116] Both cpIII and cpVIII proteins include two domains that providesignals for assembly of the mature phage particle. The first domain is asecretion signal that directs the newly synthesized protein to the hostcell membrane. The secretion signal is located at the amino terminus ofthe polypeptide and targets the polypeptide at least to the cellmembrane. The second domain is a membrane anchor domain that providessignals for association with the host cell membrane and for associationwith the phage particle during assembly. This second signal for bothcpVIII and cpIII comprises at least a hydrophobic region for spanningthe membrane.

[0117] The 50 amino acid mature gene VIII coat protein (cpVIII) issynthesized as a 73 amino acid precoat (Ito et al. (1979) PNAS76:1199-1203). cpVIII has been extensively studied as a model membraneprotein because it can integrate into lipid bilayers such as the cellmembrane in an asymmetric orientation with the acidic amino terminustoward the outside and the basic carboxy terminus toward the inside ofthe membrane. The first 23 amino acids constitute a typicalsignal-sequence which causes the nascent polypeptide to be inserted intothe inner cell membrane. An E. coli signal peptidase (SP-I) recognizesamino acids 18, 21, and 23, and, to a lesser extent, residue 22, andcuts between residues 23 and 24 of the precoat (Kuhn et al. (1985) J.Biol Chem. 260:15914-15918; and Kuhn et al. (1985) J Biol. Chem.260:15907-15913). After removal of the signal sequence, the aminoterminus of the mature coat is located on the periplasmic side of theinner membrane; the carboxy terminus is on the cytoplasmic side. About3000 copies of the mature coat protein associate side-by-side in theinner membrane.

[0118] The sequence of gene VIII is known, and the amino acid sequencecan be encoded on a synthetic gene. Mature gene VIII protein makes upthe sheath around the circular ssDNA. The gene VIII protein can be asuitable anchor protein because its location and orientation in thevirion are known (Banner et al. (1981) Nature 289:814-816). Preferably,the peptide is attached to the amino terminus of the mature M13 coatprotein to generate the phage display library. As set out above,manipulation of the concentration of both the wild-type cpVIII andAb/cpVIII fusion in an infected cell can be utilized to decrease theavidity of the display and thereby enhance the detection of highaffinity peptides directed to the target(s).

[0119] Another vehicle for displaying the peptide is by expressing it asa domain of a chimeric gene containing part or all of gene III, e.g.,encoding cpIII. When monovalent displays are required, expressing thepeptide as a fusion protein with cpII can be a preferred embodiment, asmanipulation of the ratio of wild-type cpIII to chimeric cpIII duringformation of the phage particles can be readily controlled. This geneencodes one of the minor coat proteins of M13. Genes VI, VII, and IXalso encode minor coat proteins. Each of these minor proteins is presentin about 5 copies per virion and is related to morphogenesis orinfection. In contrast, the major coat protein is present in more than2500 copies per virion. The gene VI, VII, and IX proteins are present atthe ends of the virion; these three proteins are not posttranslationallyprocessed (Rasched et al. (1986) Ann Rev. Microbiol. 41:507-541). Inparticular, the single-stranded circular phage DNA associates with aboutfive copies of the gene III protein and is then extruded through thepatch of membrane-associated coat protein in such a way that the DNA isencased in a helical sheath of protein (Webster et al. in TheSingle-Stranded DNA Phages, eds Dressler et al. (NY:CSHL Press, 1978).

[0120] Manipulation of the sequence of cpIII has demonstrated that theC-terminal 23 amino acid residue stretch of hydrophobic amino acidsnormally responsible for a membrane anchor function can be altered in avariety of ways and retain the capacity to associate with membranes. Ffphage-based expression vectors were first described in which the cpIIIamino acid residue sequence was modified by insertion of polypeptide“targets” (Parmely et al., Gene (1988) 73:305-318; and Cwirla et al.,PNAS (1990) 87:6378-6382) or an amino acid residue sequence defining asingle chain peptide domain (McCafferty et al., Science (1990)348:552-554). It has been demonstrated that insertions into gene III canresult in the prodution of novel protein domains on the virion outersurface. (Smith (1985) Science 228:1315-1317; and de la Cruz et al.(1988) J Biol. Chem. 263:4318-4322). The peptide gene may be fused togene III at the site used by Smith and by de la Cruz et al., at a codoncorresponding to another domain boundary or to a surface loop of theprotein, or to the amino terminus of the mature protein.

[0121] Generally, the successful cloning strategy utilizing a phage coatprotein, such as cpIII of filamentous phage fd, will provide expressionof a peptide chain fused to the N-terminus of a coat protein (e.g.,cpIII) and transport to the inner membrane of the host where thehydrophobic domain in the C-terminal region of the coat protein anchorsthe fusion protein in the membrane, with the N-terminus containing thepeptide chain protruding into the periplasmic space.

[0122] Similar constructions could be made with other filamentous phage.Pf3 is a well known filamentous phage that infects Pseudomonosaerugenosa cells that harbor an IncP-I plasmid. The entire genome hasbeen sequenced ((Luiten et al. (1985) J. Virol. 56:268-276) and thegenetic signals involved in replication and assembly are known (Luitenet al. (1987) DNA 6:129-137). The major coat protein of PF3 is unusualin having no signal peptide to direct its secretion. The sequence hascharged residues ASP-7, ARG-37, LYS-40, and PHE44 which is consistentwith the amino terminus being exposed. Thus, to cause a peptide toappear on the surface of Pf3, a tripartite gene can be constructed whichcomprises a signal sequence known to cause secretion in P. aerugenosa,fused in-frame to a gene fragment encoding the peptide sequence, whichis fused in-frame to DNA encoding the mature P13 coat protein.Optionally, DNA encoding a flexible linker of one to 10 amino acids isintroduced between the peptide gene fragment and the P13 coat-proteingene. This tripartite gene is introduced into P13 so that it does notinterfere with expression of any P13 genes. Once the signal sequence iscleaved off, the peptide is in the periplasm and the mature coat proteinacts as an anchor and phage-assembly signal.

[0123] b) Bacteriophage φX174

[0124] The bacteriophage φX174 is a very small icosahedral virus whichhas been thoroughly studied by genetics, biochemistry, and electronmicroscopy (see The Single Stranded DNA Phages (eds. Den hardt et al.(NY:CSHL Press, 1978)). Three gene products of φX174 are present on theoutside of the mature virion: F (capsid), G (major spike protein, 60copies per virion), and H (minor spike protein, 12 copies per virion).The G protein comprises 175 amino acids, while H comprises 328 aminoacids. The F protein interacts with the single-stranded DNA of thevirus. The proteins F, G, and H are translated from a single mRNA in theviral infected cells. As the virus is so tightly constrained becauseseveral of its genes overlap, φX174 is not typically used as a cloningvector due to the fact that it can accept very little additional DNA.However, mutations in the viral G gene (encoding the G protein) can berescued by a copy of the wild-type G gene carried on a plasmid that isexpressed in the same host cell (Chambers et al. (1982) Nuc Acid Res10:6465-6473). In one embodiment, one or more stop codons are introducedinto the G gene so that no G protein is produced from the viral genome.The variegated peptide gene library can then be fused with the nucleicacid sequence of the H gene. An amount of the viral G gene equal to thesize of peptide gene fragment is eliminated from the φX174 genome, suchthat the size of the genome is ultimately unchanged. Thus, in host cellsalso transformed with a second plasmid expressing the wild-type Gprotein, the production of viral particles from the mutant virus isrescued by the exogenous G protein source. Where it is desirable thatonly one test peptide be displayed per φX174 particle, the secondplasmid can further include one or more copies of the wild-type Hprotein gene so that a mix of H and test peptide/H proteins will bepredominated by the wild-type H upon incorporation into phage particles.

[0125] c) Large DNA Phage

[0126] Phage such as λ or T4 have much larger genomes than do M13 orφPX174, and have more complicated 3-D capsid structures than M13 orφPX174, with more coat proteins to choose from. In embodiments of theinvention whereby the test peptide library is processed and assembledinto a functional form and associates with the bacteriophage particleswithin the cytoplasm of the host cell, bacteriophage λ and derivativesthereof are examples of suitable vectors. The intracellularmorphogenesis of phage λ can potentially prevent protein domains thatordinarily contain disulfide bonds from folding correctly. However,variegated libraries expressing a population of functional peptides,which include such bonds, have been generated in λ phage. (Huse et al.(1989) Science 246:1275-1281; Mullinax et al. (1990) PNAS 87:8095-8099;and Pearson et al. (1991) PNAS 88:2432-2436). Such strategies takeadvantage of the rapid construction and efficient transformationabilities of λ phage.

[0127] When used for expression of peptide sequences (ixogenousnucleotide sequences), may be readily inserted into a λ vector. Forinstance, variegated peptide libraries can be constructed bymodification of λ ZAP II through use of the multiple cloning site of a λZAP II vector (Huse et al. supra).

[0128] ii) Bacterial Cells as Display Packages

[0129] Recombinant peptides are able to cross bacterial membranes afterthe addition of appropriate secretion signal sequences to the N-terminusof the protein (Better et al (1988) Science 240:1041-1043; and Skerra etal. (1988) Science 240:1038-1041). In addition, recombinant peptideshave been fused to outer membrane proteins for surface presentation. Forexample, one strategy for displaying peptides on bacterial cellscomprises generating a fusion protein by inserting the peptide into cellsurface exposed portions of an integral outer membrane protein (Fuchs etal. (1991) Bio/Technology 9:1370-1372). In selecting a bacterial cell toserve as the display package, any well-characterized bacterial strainwill typically be suitable, provided the bacteria may be grown inculture, engineered to display the test peptide library on its surface,and is compatible with the particular affinity selection processpracticed in the subject method. Among bacterial cells, the preferreddisplay systems include Salmonella typhirnurium, Bacillus subtilis,Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseriagonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxellabovis, and especially Escherichia coli. Many bacterial cell surfaceproteins useful in the present invention have been characterized, andworks on the localization of these proteins and the methods ofdetermining their structure include Benz et al. (1988) Ann Rev Microbiol42: 359-393; Balduyck et al. (1985) Biol Chem Hoppe-Seyler 366:9-14;Ehrmann et al (1990) PNAS 87:7574-7578; Heijne et al. (1990) ProteinEngineering 4:109-112; Ladner et al. U.S. Pat. No. 5,223,409; Ladner etal. WO88/06630; Fuchs et al. (1991) Bio/technology 9:1370-1372; andGoward et al. (1992) TIBS 18:136-140.

[0130] To further illustrate, the LamB protein of E coli is a wellunderstood surface protein that can be used to generate a variegatedlibrary of test peptides on the surface of a bacterial cell (see, forexample, Ronco et al. (1990) Biochemie 72:183-189; van der Weit et al.(1990) Vaccine 8:269-277; Charabit et al. (1988) Gene 70:181-189; andLadner U.S. Pat. No. 5,222,409). LamB of E. coli is a porin for maltoseand maltodextrin transport, and serves as the receptor for adsorption ofbacteriophages □ and K10. LamB is transported to the outer membrane if afunctional N-terminal signal sequence is present (Benson et al. (1984)PNAS 81:3830-3834). As with other cell surface proteins, LamB issynthesized with a typical signal-sequence which, is subsequentlyremoved. Thus, the variegated peptide gene library can be cloned intothe LamB gene such that the resulting library of fusion proteinscomprise a portion of LamB sufficient to anchor the protein to the cellmembrane with the test peptide fragment oriented on the extracellularside of the membrane. Secretion of the extracellular portion of thefusion protein can be facilitated by inclusion of the LamB signalsequence, or other suitable signal sequence, as the N-terminus of theprotein.

[0131] The E. coli LamB has also been expressed in functional form in S.typhimurium (Harkki et al. (1987) Mol Gen Genet 209:607-611), V.cholerae (Harkki et al. (1986) Microb Pathol 1:283-288), and K.pneumonia (Wehmeier et al. (1989) Mol Gen Genet 215:529-536), so thatone could display a population of test peptides in any of these speciesas a fusion to E. coli LamB. Moreover, K. pneumonia expresses amaltoporin similar to LamB which could also be used. In P. aeruginosa,the D1 protein (a homologue of LamB) can be used (Trias et al. (1988)Biochem Biophys Acta 938:493-496). Similarly, other bacterial surfaceproteins, such as PAL, OmpA, OmpC, OmpF, PhoE, pilin, BtuB, FepA, FhuA,lutA, FecA and FhuE, may be used in place of LamB as a portion of thedisplay means in a bacterial cell.

[0132] In another exemplary embodiment, the fusion protein can bederived using the FliTrx™ Random Peptide Display Library (Invitrogen).That library is a diverse population of random dodecapeptides insertedwithin the thioredoxin active-site loop inside the dispensable region ofthe bacterial flagellin gene (fliC). The resultant recombinant fusionprotein (FLITRX) is exported and assembled into partially functionalflagella on the bacterial cell surface, displaying the random peptidelibrary.

[0133] Peptides are fused in the middle of thioredoxin, therefore, boththeir N- and C-termini are anchored by thioredoxin's tertiary structure.This results in the display of a constrained peptide. By contrast, phagedisplay proteins are fused to the N-terminus of phage coat proteins inan unconstrained manner. The unconstrained molecules possess manydegrees of conformational freedom which may result in the lack of properinteraction with the target molecule. Without proper interaction, manypotential protein-protein interactions may be missed.

[0134] Moreover, phage display is limited by the low expression levelsof bacteriophage coat proteins. FliTrx™ and similar methods can overcomethis limitation by using a strong promoter to drive expression of thetest peptide fusions that are displayed as multiple copies.

[0135] According to the present invention, it is contemplated that theFliTrx vector can be modified to provide, similar to the illustratedvectors of the attached figures, a vector which is differentiallyspliced in mammalian cells to yield a secreted, soluble test peptide.

[0136] iii) Bacterial Spores as Display Packages

[0137] Bacterial spores also have desirable properties as displaypackage candidates in the subject method. For example, spores are muchmore resistant than vegetative bacterial cells or phage to chemical andphysical agents, and hence permit the use of a great variety of affinityselection conditions. Also, Bacillus spores neither actively metabolizenor alter the proteins on their surface. However, spores have thedisadvantage that the molecular mechanisms that trigger sporulation areless well worked out than is the formation of M13 or the export ofprotein to the outer membrane of E. coli, though such a limitation isnot a serious detractant from their use in the present invention.

[0138] Bacteria of the genus Bacillus form endospores that are extremelyresistant to damage by heat, radiation, desiccation, and toxic chemicals(reviewed by Losick et al. (1986) Ann Rev Genet 20:625-669). Thisphenomenon is attributed to extensive intermolecular cross-linking ofthe coat proteins. In certain embodiments of the subject method, such asthose which include relatively harsh affinity separation steps, Bacillusspores can be the preferred display package. Endospores from the genusBacillus are more stable than are, for example, exospores fromStreptomyces. Moreover, Bacillus subtilis forms spores in 4 to 6 hours,whereas Streptomyces species may require days or weeks to sporulate. Inaddition, genetic knowledge and manipulation is much more developed forB. subtilis than for other spore-forming bacteria.

[0139] Viable spores that differ only slightly from wild-type areproduced in B. subtilis even if any one of four coat proteins is missing(Donovan et al. (1987) J Mol Biol 196:1-10). Moreover, plasmid DNA iscommonly included in spores, and plasmid encoded proteins have beenobserved on the surface of Bacillus spores (Debro et al. (1986) JBacteriol 165:258-268). Thus, it can be possible during sporulation toexpress a gene encoding a chimeric coat protein comprising a peptide ofthe variegated gene library, without interfering materially with sporeformation.

[0140] To illustrate, several polypeptide components of B. subtilisspore coat (Donovan et al. (1987) J Mol Biol 196:1-10) have beencharacterized. The sequences of two complete coat proteins andamino-terminal fragments of two others have been determined. Fusion ofthe test peptide sequence to cotC or cotD fragments is likely to causethe peptide to appear on the spore surface. The genes of each of thesespore coat proteins are preferred as neither cotC or cotD arepost-translationally modified (see Ladner et al. U.S. Pat. No.5,223,409).

[0141] iv) Selecting Peptides from the Display Mode

[0142] Upon expression, the variegated peptide display is subjected toaffinity enrichment in order to select for test peptides which bindpreselected targets. The term “affinity separation” or “affinityenrichment” includes, but is not limited to: (1) affinity chromatographyutilizing immobilized targets, (2) immunoprecipitation using solubletargets, (3) fluorescence activated cell sorting, (4) agglutination, and(5) plaque lifts. In each embodiment, the library of display packagesare ultimately separated based on the ability of the associated testpeptide to bind the target of interest. See, for example, the Ladner etal. U.S. Pat. No. 5,223,409; the Kang et al. International PublicationNo. WO 92/18619; the Dower et al. International Publication No. WO91/17271; the Winter et al. International Publication WO 92/20791; theMarkland et al. International Publication No. WO 92/15679; the Breitlinget al. International Publication WO 93/01288; the McCafferty et al.International Publication No. WO 92/01047; the Garrard et al.International Publication No. WO 92/09690; and the Ladner et al.International Publication No. WO 90/02809. In most preferredembodiments, the display library will be pre-enriched for peptidesspecific for the target by first contacting the display library with anynegative controls or other targets for which differential binding by thetest peptide is desired. Subsequently, the non-binding fraction fromthat pre-treatment step is contacted with the target and peptides fromthe display which are able to specifically bind the target are isolated.

[0143] With respect to affinity chromatography, it will be generallyunderstood by those skilled in the art that a great number ofchromatography techniques can be adapted for use in the presentinvention, ranging from column chromatography to batch elution, andincluding ELISA and biopanning techniques. Typically, where the targetis a component of a cell, rather than a whole cell, the target isimmobilized on an insoluble carrier, such as sepharose or polyacrylamidebeads, or, alternatively, the wells of a microtitre plate. As describedbelow, in instances where no purified source of the target is readilyavailable, such as the case with many cell surface receptors, the cellson which the target is displayed may serve as the insoluble matrixcarrier.

[0144] The population of display packages is applied to the affinitymatrix under conditions compatible with the binding of the test peptideto a target. The population is then fractionated by washing with asolute that does not greatly effect specific binding of peptides to thetarget, but which substantially disrupts any non-specific binding of thedisplay package to the target or matrix. A certain degree of control canbe exerted over the binding characteristics of the peptides recoveredfrom the display library by adjusting the conditions of the bindingincubation and subsequent washing. The temperature, pH, ionic strength,divalent cation concentration, and the volume and duration of thewashing can select for peptides within a particular range of affinityand specificity. Selection based on slow dissociation rate, which isusually predictive of high affinity, is a very practical route. This maybe done either by continued incubation in the presence of a saturatingamount of free hapten (if available), or by increasing the volume,number, and length of the washes. In each case, the rebinding ofdissociated peptide-display package is prevented, and with increasingtime, peptide-display packages of higher and higher affinity arerecovered. Moreover, additional modifications of the binding and washingprocedures may be applied to find peptides with special characteristics.The affinities of some peptides are dependent on ionic strength orcation concentration. This is a useful characteristic for peptides to beused in affinity purification of various proteins when gentle conditionsfor removing the protein from the peptide are required. Specificexamples are peptides which depend on Ca⁺⁺ for binding activity andwhich lose or gain binding affinity in the presence of EGTA or othermetal chelating agent. Such peptides may be identified in therecombinant peptide library by a double screening technique isolatingfirst those that bind the target in the presence of Ca⁺⁺, and bysubsequently identifying those in this group that fail to bind in thepresence of EGTA.

[0145] After “washing” to remove non-specifically bound displaypackages, when desired, specifically bound display packages can beeluted by either specific desorption (using excess target) ornon-specific desorption (using pH, polarity reducing agents, orchaotropic agents). In preferred embodiments, the elution protocol doesnot kill the organism used as the display package such that the enrichedpopulation of display packages can be further amplified by reproduction.The list of potential eluants includes salts (such as those in which oneof the counter ions is Na⁺, NH₄ ⁺, Rb⁺, SO₄ ²⁻, H₂PO₄, citrate, K⁺, Li⁺,Cs⁺, HSO₄ ⁻, CO₃ ²⁻, Ca²⁺, Sr²⁺, Cl⁻, PO₄ ²⁻, HCO₃ ⁻, Mg₂ ⁺, Ba₂ ⁺, Br⁻,HPO₄ ²⁻, or acetate), acid, heat, and, when available, soluble forms ofthe target target (or analogs thereof). Because bacteria continue tometabolize during the affinity separation step and are generally moresusceptible to damage by harsh conditions, the choice of buffercomponents (especially eluates) can be more restricted when the displaypackage is a bacteria rather than for phage or spores. Neutral solutes,such as ethanol, acetone, ether, or urea, are examples of other agentsuseful for eluting the bound display packages.

[0146] In preferred embodiments, affinity enriched display packages areiteratively amplified and subjected to further rounds of affinityseparation until enrichment of the desired binding activity is detected.In certain embodiments, the specifically bound display packages,especially bacterial cells, need not be eluted per se, but rather, thematrix bound display packages can be used directly to inoculate asuitable growth media for amplification.

[0147] Where the display package is a phage particle, the fusion proteingenerated with the coat protein can interfere substantially with thesubsequent amplification of eluted phage particles, particularly inembodiments wherein the cpIII protein is used as the display anchor.Even though present in only one of the 5-6 tail fibers, some peptideconstructs because of their size and/or sequence, may cause severedefects in the infectivity of their carrier phage. This causes a loss ofphage from the population during reinfection and amplification followingeach cycle of panning. In one embodiment, the peptide can be derived onthe surface of the display package so as to be susceptible toproteolytic cleavage which severs the covalent linkage of at least thetarget binding sites of the displayed peptide from the remainingpackage. For instance, where the cpIII coat protein of M13 is employed,such a strategy can be used to obtain infectious phage by treatment withan enzyme which cleaves between the test peptide portion and cpIIIportion of a tail fiber fusion protein (e.g. such as the use of anenterokinase cleavage recognition sequence).

[0148] To further minimize problems associated with defectiveinfectivity, DNA prepared from the eluted phage can be transformed intohost cells by electroporation or well known chemical means. The cellsare cultivated for a period of time sufficient for marker expression,and selection is applied as typically done for DNA transformation. Thecolonies are amplified, and phage harvested for a subsequent round(s) ofpanning.

[0149] After isolation of display packages which encode peptides havinga desired binding specificity for the target, the test peptides for eachof the purified display packages can be tested for biological activityin the secretion mode of the subject method.

[0150] B. Secretion Mode

[0151] In the “secretion mode,” the combinatorial peptide library, whichhas been enriched in the display mode, is transfected into and expressedby eukaryotic cells. In this mode, the test peptides are secreted by thehost cells and screened for biological activity.

[0152] In preferred embodiments, and illustrated in the drawings, thesubject vectors are constructed to include eukaryotic splice sites suchthat, in the mature MRNA, elements required for the display mode inprokaryotic cells are spliced out—at least those elements which wouldinterfere with the secretion mode. A variety of naturally andnon-naturally occurring splice sites are available in the art and can beselected for, e.g., optimization in particular eukaryotic cellsselected.

[0153] In preferred embodiments, the vectors of the subject inventionare used to transfect a cell that can be co-cultured with a target cell.A biologically active protein secreted by the cells expressing thecombinatorial library will diffuse to neighboring target cells andinduce a particular biological response, such as to illustrate,proliferation or differentiation, or activation of a signal transductionpathway which is directly detected by other phenotypic criteria. Thepattern of detection of biological activity will resemble a gradientfunction, and will allow the isolation (generally after severalrepetitive rounds of selection) of cells producing peptides havingcertain activity in the assay. Likewise, antagonists of a given factorcan be selected in similar fashion by the ability of the cell producinga functional antagonist to protect neighboring cells from the effect ofexogenous factor added to the culture media.

[0154] To further illustrate, target cells are cultured in 24-wellmicrotitre plates. Other cells are transfected with the combinatorialpeptide library, recovered after the display mode step, and cultured incell culture inserts (e.g. Collaborative Biomedical Products, Catalog#40446) that are able to fit into the wells of the microtitre plate. Thecell culture inserts are placed in the wells such that recombinant testpeptides secreted by the cells in the insert can diffuse through theporous bottom of the insert and contact the target cells in themicrotitre plate wells. After a period of time sufficient for a secretedtest peptide to produce a measurable response in the target cells, theinserts are removed and the effect of the peptides on the target cellsdetermined. For example, where the target cell is a neural crest celland the activity desired from the test peptides is the induction ofneuronal differentiation, then fluorescently-labeled antibodies specificfor Islet-1 or other neuronal markers can be used to score for inductionin the target cells as indicative of a functional neurotrophic peptidein that well. Cells from the inserts corresponding to wells which scorepositive for activity can be split and re-cultured on several inserts,the process being repeated until the active peptide is identified.

[0155] When screening for bioactivity of test peptides, intracellularsecond messenger generation can be measured directly. For instance, avariety of intracellular effectors have been identified as beingreceptor- or ion channel-regulated, including adenylyl cyclase, cyclicGMP, phosphodiesterases, phosphoinositidases, phosphoinositol kinases,and phospholipases, as well as a variety of ions.

[0156] In one embodiment, the GTPase enzymatic activity by G proteinscan be measured in plasma membrane preparations by determining thebreakdown of γ³²P GTP using techniques that are known in the art (Forexample, see Signal Transduction: A Practical Approach. G. Milligan, Ed.Oxford University Press, Oxford England). When receptors that modulatecAMP are tested, it will be possible to use standard techniques for cAMPdetection, such as competitive assays which quantitate [³H]cAMP in thepresence of unlabelled cAMP.

[0157] Certain receptors and ion channels stimulate the activity ofphospholipase C which stimulates the breakdown of phosphatidylinositol4,5, bisphosphate to 1,4,5-IP3 (which mobilizes intracellular Ca++) anddiacylglycerol (DAG) (which activates protein kinase C). Inositol lipidscan be extracted and analyzed using standard lipid extractiontechniques. DAG can also be measured using thin-layer chromatography.Water soluble derivatives of all three inositol lipids (IP1, IP2, IP3)can also be quantitated using radiolabelling techniques or HPLC.

[0158] The other product of PIP2 breakdown, DAG can also be producedfrom phosphatidyl choline. The breakdown of this phospholipid inresponse to receptor-mediated signaling can also be measured using avariety of radiolabelling techniques.

[0159] The activation of phospholipase A2 can easily be quantitatedusing known techniques, including, for example, the generation ofarachadonate in the cell.

[0160] In various cells, e.g., mammalian cells, specific proteases areinduced or activated in each of several arms of divergent signalingpathways. These may be independently monitored by following their uniqueactivities with substrates specific for each protease.

[0161] In the case of certain receptors and ion channels, it may bedesirable to screen for changes in cellular phosphorylation. Such assayformats may be useful when, for example, the assay is designed to detectan agonist or antagonist of a receptor kinase or phosphatase. Forexample, immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci.USA 81:7426-7430) using anti-phosphotyrosine, anti-phosphoserine orabti-phosphothreonine antibodies. In addition, tests for phosphorylationcould be also useful when the receptor itself may not be a kinase, butactivates protein kinases or phosphatase that function downstream in thesignal transduction pathway.

[0162] One such cascade is the MAP kinase pathway that appears tomediate both mitogenic, differentiation and stress responses indifferent cell types. Stimulation of growth factor receptors results inRas activation followed by the sequential activation of c-Raf, MEK, andp44 and p42 MAP kinases (ERKI and ERK2). Activated MAP kinase thenphosphorylates many key regulatory proteins, including p90RSK and Elk-1that are phosphorylated when MAP kinase translocates to the nucleus.Homologous pathways exist in mammalian and yeast cells. For instance, anessential part of the S. cerevisiae pheromone signaling pathway iscomprised of a protein kinase cascade composed of the products of theSTE11, STE7, and FUS3/KSS1 senes (the latter pair are distinct andfunctionally redundant). Accordingly, phosphorylation and/or activationof members of this kinase cascade can be detected and used to quantitatereceptor engagement. Phosphotyrosine specific antibodies are availableto measure increases in tyrosine phosphorylation and phospho-specificantibodies are commercially available (New England Biolabs, Beverly,Mass.).

[0163] In yet another embodiment, the signal transduction pathway ofinterest may upregulate expression or otherwise activate an enzyme whichis capable of modifying a substrate which can be added to the cell. Thesignal can be detected by using a detectable substrate, in which caselose of the substrate signal is monitored, or alternatively, by using asubstrate which produces a detectable product. In preferred embodiments,the conversion of the substrate to product by the activated enzymeproduces a detectable change in optical characteristics of the testcell, e.g., the substrate and/or product is chromogenically orfluorogenically active. In an illustrative embodiment the signaltransduction pathway causes a change in the activity of a proteolyticenzyme, altering the rate at which it cleaves a substrate peptide (orsimply activates the enzyme towards the substrate). The peptide includesa fluorogenic donor radical, e.g., a fluorescence emitting radical, andan acceptor radical, e.g., an aromatic radical which absorbs thefluorescence energy of the fluorogenic donor radical when the acceptorradical and the fluorogenic donor radical are covalently held in closeproximity. See, for example, U.S. Ser. No. 5,527,681, 5,506,115,5,429,766, 5,424,186, and 5,316,691; and Capobianco et al. (1992) AnalBiochem 204:96-102. For example, the substrate peptide has afluorescence donor group such as 1-aminobenzoic acid (anthranilic acidor ABZ) or aminomethylcoumarin (AMC) located at one position on thepeptide and a fluorescence quencher group, such as lucifer yellow,methyl red or nitrobenzo-2-oxo-1,3-diazole (NBD), at a differentposition near the distal end of the peptide. A cleavage site for theactivated enzyme will be disposed between each of the sites for thedonor and acceptor groups. The intramolecular resonance energy transferfrom the fluorescence donor molecule to the quencher will quench thefluorescence of the donor molecule when the two are sufficientlyproximate in space, e.g., when the peptide is intact. Upon cleavage ofthe peptide, however, the quencher is separated from the donor group,leaving behind a fluorescent fragment. Thus, activation of the enzymeresults in cleavage of the detection peptide, and dequenching of thefluorescent group.

[0164] In. still other embodiments, the detectable signal can beproduced by use of enzymes or chromogenic/fluorscent probes whoseactivities are dependent on. the concentration of a second messanger,e.g., such as calcium, hydrolysis products of inositol phosphate, cAMP,etc. For example ? the mobilization of intracellular calcium or theinflux of calcium from outside the cell can be measured using standardtechniques. The choice of the appropriate calcium indicator,fluorescent, bioluminescent, metallochromic, or Ca++-sensitivemicroelectrodes depends on the cell type and the magnitude and timeconstant of the event under study (Borle (1990) Environ Health Perspect84:45-56). As an exemplary method of Ca++ detection, cells could beloaded with the Ca++ sensitive fluorescent dye fura-2 or indo-1, usingstandard methods, and any change in Ca++ measured using a fluorometer.

[0165] As certain embodiments described above suggest, in addition todirectly measuring second messenger production, the signal transductionactivity of a receptor or ion channel pathway can be measured bydetection of a transcription product, e.g., by detectingreceptor/channel-mediated transcriptional activation (or repression) ofa gene(s). Detection of the transcription product includes detecting thegene transcript, detecting the product directly (e.g., by immunoassay)or detecting an activity of the protein (e.g., such as an enzymaticactivity or chromogenic/fluorogenic activity); each of which isgenerally referred to herein as a means for detecting expression of theindicator gene. The indicator gene may be an unmodified endogenous geneof the host cell, a modified endogenous gene, or a part of a completelyheterologous construct, e.g., as part of a reporter gene construct.

[0166] In one embodiment, the indicator gene is an unmodified endogenousgene. For example, the instant method can rely on detecting thetranscriptional level of such endogenous genes as the c-fos gene (e.g.,in mammalian cells) or the Bar1 or Fus1 genes (e.g., in yeast cells) inresponse to such signal transduction pathways as originating from Gprotein coupled receptors.

[0167] In certain instances, it may be desirable to increase the levelof transcriptional activation of the endogenous indicator gene by thesignal pathway in order to, for example, improve the signal-to-noise ofthe test system, or to adjust the level of response to a level suitablefor a particular detection technique. In one embodiment, thetranscriptional activation ability of the signal pathway can beamplified by the overexpression of one or more of the proteins involvedin the intracellular signal cascade, particularly enzymes involved inthe pathway. For example, increased expression of Jun kinases (JNKs) canpotentiate the level of transcriptional activation by a signal in anMEKK/JNKK pathway. Likewise, overexpression of one or more signaltransduction proteins in the yeast pheromone pathway can increase thelevel of Fusl and/or Bar1 expression. This approach can, of course, alsobe used to potentiate the level of transcription of a heterologousreporter gene as well.

[0168] In other embodiments, the sensitivity of an endogenous indicatorgene can be enhanced by manipulating the promoter sequence at thenatural locus for the indicator gene. Such manipulation may range frompoint mutations to the endogenous regulatory elements to grossreplacement of all or substantial portions of the regulatory elements:In general, manipulation of the genomic sequence for the indicator genecan be carried out using techniques known in the art, includinghomologous recombination.

[0169] In another exemplary embodiment, the promoter (or othertranscriptional regulatory sequences) of the endogenous gene can be“switched out” with a heterologous promoter sequence, e.g., to form achimeric gene at the indicator gene locus. Again, using such techniquesas homologous recombination, the regulatory sequence can be so alteredat the genomic locus of the indicator gene.

[0170] In still another embodiment, a heterologous reporter geneconstruct can be used to provide the function of an indicator gene.Reporter gene constructs are prepared by operatively linking a reportergene with at least one transcriptional regulatory element. If only onetranscriptional regulatory element is included it must be a regulatablepromoter. At least one the selected transcriptional regulatory elementsmust be indirectly or directly regulated by the activity of the selectedcell-surface receptor whereby activity of the receptor can be monitoredvia transcription of the reporter genes.

[0171] Many reporter genes and transcriptional regulatory elements areknown to those of skill in the art and others may be identified orsynthesized by methods known to those of skill in the art.

[0172] Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864-869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667);alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238,Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secretedalkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol.216:362-368); β-lactamase or GST.

[0173] Transcriptional control elements for use in the reporter geneconstructs, or for modifying the genomic locus of an indicator geneinclude, but are not limited to, promoters, enhancers, and repressor andactivator binding sites. Suitable transcriptional regulatory elementsmay be derived from the transcriptional regulatory regions of geneswhose expression is rapidly induced, generally within minutes, ofcontact between the cell surface protein and the effector protein thatmodulates the activity of the cell surface protein. Examples of suchgenes include, but are not limited to, the immediate early genes (see,Sheng et al. (1990) Neuron 4: 477-485), such as c-fos. Immediate earlygenes are genes that are rapidly induced upon binding of a ligand to acell surface protein. The transcriptional control elements that arepreferred for use in the gene constructs include transcriptional controlelements from immediate early genes, elements derived from other genesthat exhibit some or all of the characteristics of the immediate earlygenes, or synthetic elements that are constructed such that genes inoperative linkage therewith exhibit such characteristics. Thecharacteristics of preferred genes from which the transcriptionalcontrol elements are derived include, but are not limited to, low orundetectable expression in quiescent cells, rapid induction at thetranscriptional level within minutes of extracellular simulation,induction that is transient and independent of new protein synthesis,subsequent shut-off of transcription requires new protein synthesis, andmRNAs transcribed from these genes have a short half-life. It is notnecessary for all of these properties to be present.

[0174] Other promoters and transcriptional control elements, in additionto those described above, include the vasoactive intestinal peptide(VIP) gene promoter (cAMP responsive; Fink et al. (1988), Proc. Natl.Acad. Sci. 85:6662-6666); the somatostatin gene promoter (cAMPresponsive; Montminy et al. (1986), Proc. Natl. Acad. Sci.8.3:6682-6686); the proenkephalin promoter (responsive to cAMP,nicotinic agonists, and phorbol esters; Comb et al. (1986), Nature323:353-356); the phosphoenolpyruvate carboxy-kinase gene promoter (cAMPresponsive; Short et al. (1986), J. Biol. Chem. 261:9721-9726); theNGFI-A gene promoter (responsive to NGF, cAMP, and serum; Changelian etal. (1989). Proc. Natl. Acad. Sci. 86:377-381); and others that may beknown to or prepared by those of skill in the art.

[0175] In the case of receptors which modulate cyclic AMP, atranscriptional based readout can be constructed using the cyclic AMPresponse element binding protein, CREB, which is a transcription factorwhose activity is regulated by phosphorylation at a particular serine(S133). When this serine residue is phosphorylated, CREB binds to arecognition sequence known as a CRE (cAMP Responsive Element) found tothe 5′ of promotors known to be responsive to elevated cAMP levels. Uponbinding of phosphorylated CREB to a CRE, transcription from thispromoter is increased.

[0176] Phosphorylation of CREB is seen in response to both increasedcAMP levels and increased intracellular Ca levels. Increased cAMP levelsresult in activation of PKA, which in :turn phosphorylates CREB andleads to binding to CRE and transcriptional activation. Increasedintracellular calcium levels results in activations. ofcalcium/calmodulin responsive kinase II (CaM kinase II). Phosphorylationof CREB by CaM kinase II is effectively the same as phosphorylation ofCREB by PKA, and results in transcriptional activation of CRE containingpromotors.

[0177] Therefore, a transcriptionally-based readout can be constructedin cells containing a reporter gene whose expression is driven by abasal promoter containing one or more CRE. Changes in the intracellularconcentration of Ca++ (a result of alterations in the activity of thereceptor upon engagement with a ligand) will result in changes in thelevel of expression of the reporter gene if: a) CREB is alsoco-expressed in the cell, and b) either an endogenous or heterologousCaM kinase phosphorylates CREB in response to increases in calcium or ifan exogenously expressed CaM kinase II is present in the same cell. Inother words, stimulation of PLC activity may result in phosphorylationof CREB and increased transcription from the CRE-construct, whileinhibition of PLC activity may result in decreased transcription fromthe CRE-responsive construct.

[0178] As described in Bonni et al. (1993) Science 262:1575-1579, theobservation that CNTF treatment of SK-N-MC cells leads to the enhancedinteraction of STAT/p91 and STAT related proteins with specific DNAsequences suggested that these proteins might be key regulators ofchanges in gene expression that are triggered by CNTF. Consistent withthis possibility is the finding that DNA sequence elements similar tothe consensus DNA sequence required for STAT/p91 binding are presentupstream of a number of genes previously found to be induced by CNTF(e.g., Human c-fos, Mouse c-fos, Mouse tis11, Rat junB, Rat SOD-1, andCNTF). Those authors demonstrated the ability of STAT/p91 binding sitesto confer CNTF responsiveness to a non-responsive reporter gene.Accordingly, a reporter construct for use in the present invention fordetecting signal transduction through STAT proteins, such as fromcytokine receptors, can be generated by using -71 to +109 of the mousec-fos gene fused to the bacterial chloramphenicol acetyltransferase gene(−71fosCAT) or other detectable marker gene. Induction by a cytokinereceptor induces the tyrosine phosphorylation of STAT and STAT-relatedproteins, with subsequent translocation and binding of these proteins tothe STAT-RE. This then leads to activation of transcription of genescontaining this DNA element within their promoters.

[0179] In preferred embodiments, the reporter gene is a gene whoseexpression causes a phenotypic change which is screenable or selectable.If the change is selectable, the phenotypic change creates a differencein the growth or survival rate between cells which express the reportergene and those which do not. If the change is screenable, the phenotypechange creates a difference in some detectable characteristic of thecells, by which the cells which express the marker may be distinguishedfrom those which do not. Selection is preferable to screening in that itcan provide a means for amplifying from the cell culture those cellswhich express a test polypeptide which is a receptor effector.

[0180] The marker gene is coupled to the receptor signaling pathway sothat expression of the marker gene is dependent on activation of thereceptor. This coupling may be achieved by operably linking the markergene to a receptor-responsive promoter. The term “receptor-responsivepromoter”, indicates a promoter which is regulated by some product ofthe target receptor's signal transduction pathway.

[0181] Alternatively, the promoter may be one which is repressed by thereceptor pathway, thereby preventing expression of a product which isdeleterious to the cell. With a receptor repressed promoter, one screensfor agonists by linking the promoter to a deleterious gene, and forantagonists, by linking it to a beneficial gene. Repression may beachieved by operably linking a receptor- induced promoter to a geneencoding mRNA which is antisense to at least a portion of the mRNAencoded by the marker gene (whether in the coding or flanking regions),so as to inhibit translation of that MRNA. Repression may also beobtained by linking a receptor-induced promoter to a gene encoding a DNAbinding repressor protein, and incorporating a suitable operator siteinto the promoter or other suitable region of the marker gene.

[0182] The marker gene may also be a screenable gene. The screenedcharacteristic may be a change in cell morphology, metabolism or otherscreenable features. Suitable markers include □-galactosidase (Xgal,C₁₂FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkalinephosphatase, horseradish peroxidase, exo-glucanase (product of yeastexb1 gene; nonessential, secreted); luciferase; bacterial greenfluorescent protein; (human placental) secreted alkaline phosphatase(SEAP); and chloramphenicol transferase (CAT). Some of the above can beengineered so that they are secreted (although not □-galactosidase). Apreferred screenable marker gene is beta-galactosidase; yeast cellsexpressing the enzyme convert the colorless substrate Xgal into a bluepigment. Again, the promoter may be receptor-induced orreceptor-inhibited.

[0183] In certain assays it may be desirable to use changes in growth inthe screening procedure. For example, one of the consequences ofactivation of the pheromone signal pathway in wild-type yeast is growtharrest. If one is testing for an antagonist of a G protein-coupledreceptor, such as a human receptor engineered into a yeast cell, thisnormal response of growth arrest can be used to select cells in whichthe pheromone response pathway is inhibited. That is, cells exposed to atest compound will be growth arrested if the compound is an agonist, butwill grow normally if the compound is neutral or an antagonist. Thus,the growth arrest response can be used to advantage to discovercompounds that function as agonists or antagonists. Moreover, the effectof growth arrest can provide a selective advantage in the presence of anagent which is cytotoxic to mitotic cells. For example, during thegrowth arrest window, the cytotoxic agent is added to the culture. Cellswhich proceed through the cell-cycle, e.g., which are not growtharrested, will be killed. At some time after the addition of thecytotoxic agent, it can be washed from the culture, and surviving cellspermited to proceed with proliferation. Cells which were arrested by thetest compound will be enriched in the surviving population.

[0184] However, in certain embodiments the growth arrest consequent toactivation of the pheromone response pathway is an undesirable effectsince cells that bind agonists stop growing while surrounding cells thatfail to bind peptides will continue to grow. The cells of interest,then, will be overgrown or their detection obscured by the backgroundcells, confounding identification of agonistic peptides. To overcomethis problem the present invention teaches engineering the cell suchthat: 1) growth arrest does not occur as a result of exogenous signalpathway activation (e.g., by inactivating the FARI gene); and/or 2) aselective growth advantage is conferred by activating the pathway (e.g.,by transforming an auxotrophic mutant with a HIS3 gene under the controlof a pheromone-responsive promoter, and applying selective conditions).

[0185] It is, of course, desirable that the exogenous receptor beexposed on a continuing basis to the peptides. Unfortunately, this islikely to result in desensitization of the pheromone pathway to thestimulus. For example, the mating signal transduction pahtway is knownto become desensitized by several mechanisms including pheromonedegradation and modification of the function of the receptor, Gproteins,s and/or downstream elements of the pheromone signaltransduction by the products of the SST2, STE50, AFRI (Konopka, J. B.(1993) Mol. Cell. Biol. 13:6876-6888) and SGV1, MSG5, and SIG1 genes.Selected mutations in these genes can lead to hypersensitivity topheromone and an inability to adapt to the presence of pheromone. Forexample, introduction of mutations that interfere with function intostrains expressing heterologous G protein-coupled receptors constitutesa significant improvement on wild type strains and enables thedevelopment of extremely sensitive bioassays for compounds that interactwith the receptors. Other mutations e.g. STE50, sgv1, bar1,ste2,ste3,pik1, msg5, sig1, and aft1, have the similar effect ofincreasing the sensitivity of the bioassay. Thus desensitization may beavoided by mutating (which may include deleting) the SST2 gene so thatit no longer produces a functional protein, or by mutating one of theother genes listed above.

[0186] If the endogenous homolog of the receptor is produced by theyeast cell, the assay will not be able to distinguish between peptideswhich interact with the endogenous receptor and those which interactwith the exogenous receptor. It is therefore desirable that theendogenous gene be deleted or otherwise rendered nonfunctional.

[0187] Suitable host cells for generating the target cells of subjectassay include prokaryotes, yeast, or higher eukaryotic cells, includingplant and animal cells, especially mammalian cells. Prokaryotes includegram negative or gram positive organisms. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman (1981) Cell 23:175) CV-1 cells (ATCC CCL 70), L cells,C127, 3T3, Chinese hamster ovary (CHO), HeLa, HEK-293, SWISS 3T3, andBHK cell lines.

[0188] If yeast cells are used, the yeast may be of any species whichare cultivable and in which an exogenous receptor can be made to engagethe appropriate signal transduction machinery of the host cell. Suitablespecies include Kluyverei lactis, Schizosaccharomyces pombe, andUstilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeastwhich can be used in practicing the present invention. are Neurosporacrassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris,Candida tropicalis, and Hansenula polymorpha. The term “yeast”, as usedherein, includes not only yeast in a strictly taxonomic sense, i.e.,unicellular organisms, but also yeast-like multicellular fungi orfilamentous fungi.

[0189] The choice of appropriate host cell will also be influenced bythe choice of detection signal. For instance, reporter constructs, asdescribed below, can provide a selectable or screenable trait upontranscriptional activation (or inactivation) in response to a signaltransduction pathway coupled to the target receptor. The reporter genemay be an unmodified gene already in the host cell pathway. It may be ahost cell gene that has been operably linked to a “receptor-responsive”promoter. Alternatively, it may be a heterologous gene (e.g., a“reporter gene construct”) that has been so linked. Suitable genes andpromoters are discussed below. In other embodiments, second messengergeneration can be measured directly in the detection step, such asmobilization of intracellular calcium or phospholipid metabolism arequantitated. In yet other embodiments indicator genes can be used todetect receptor-mediated signaling.

[0190] Accordingly, it will be understood that to achieve selection orscreening, the host cell must have an appropriate phenotype. Forexample, generating a pheromone-responsive chimeric HIS3 gene in a yeastthat has a wild-type HIS3 gene would frustrate genetic selection. Thus,to achieve nutritional selection, an auxotrophic strain is wanted.

[0191] A variety of complementations for use in the subject assay can beconstructed. Indeed, many yeast genetic complementation with mammaliansignal transduction proteins have been described in the art. Forexample, Mosteller et al. (1994) Mol Cell Biol 14:1104-12 demonstratesthat human Ras proteins can complement loss of ras mutations in S.cerevisiae. Moreover, Toda et al. (1986) Princess Takamatsu Symp 17:253-60 have shown that human ras proteins can complement the loss of RAS1 and RAS2 proteins in yeast, and hence are functionally homologous.Both human and yeast RAS proteins can stimulate the magnesium andguanine nucleotide-dependent adenylate cyclase activity present in yeastmembranes. Ballester et al. (1989) Cell 59: 681-6 describe a vector toexpress the mammalian GAP protein in the yeast S. cerevisiae. Whenexpressed in yeast, GAP inhibits the function of the human ras protein,and complements the loss of IRA1. IRA1 is a yeast gene that encodes aprotein with homology to GAP and acts upstream of RAS. Mammalian GAP cantherefore function in yeast and interact with yeast RAS. Wei et al.(1994) Gene 151: 279-84 describes that a human Ras-specific guaninenucleotide-exchange factor, Cdc25GEF, can complement the loss of CDC25function in S. cerevisiae. Martegani et al. (1992) EMBO J 11: 2151-7describe the cloning by functional complementation of a mouse cDNAencoding a homolog of CDC25, a Saccharomyces cerevisiae RAS activator.Vojtek et al. (1993) J Cell Sci 105: 777-85 and Matviw et al. (1992) MolCell Biol 12: 5033-40 describe how a mouse CAP protein, e.g., anadenylyl cyclase associated protein associated with ras-mediated signaltransduction, can complements defects in S. cerevisiae. Papasavvas etal. (1992) Biochem Biophys Res Commun 184:1378-85 also suggest thatinactivated yeast adenyl cyclase can be complemented by a mammalianadenyl cyclase gene. Hughes et al. (1993) Nature 364: 349-52 describethe complementation of byr1 in fission yeast by mammalian MAP kinasekinase (MEK). Parissenti et al. (1993) Mol Cell Endocrinol 98: 9-16describes the reconstitution of bovine protein kinase C (PKC) in yeast.The Ca(2+)− and phospholipid-dependent Ser/Thr kinase PKC playsimportant roles in the transduction of cellular signals in mammaliancells. Marcus et al. (1995) PNAS 92: 6180-4 suggests the complementationof shkl null mutations in S. pombe by the either the structurallyrelated S. cerevisiae Ste20 or mammalian p65PAK protein kinases.

[0192] “Inactivation”, with respect to genes of the host cell, meansthat production of a functional gene product is prevented or inhibited.Inactivation may be achieved by deletion of the gene, mutation of thepromoter so that expression does not occur, or mutation of the codingsequence so that the gene product is inactive. Inactivation may bepartial or total.

[0193] “Complementation”, with respect to genes of the host cell, meansthat at least partial function of inactivated gene of the host cell issupplied by an exogenous nucleic acid. For instance, yeast cells can be“mammalianized”, and even “humanized”, by complementation of receptorand signal transduction proteins with mammalian homologs. To illustrate,inactivation of a yeast Byr2/Ste11 gene can be complemented byexpression of a human MEKK gene.

[0194] C. Generations of Peptide Libraries

[0195] The variegated peptide libraries of the subject method can begenerated by any of a number of methods, and, though not limited by,preferably exploit recent trends in the preparation of chemicallibraries. For instance, chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic genes then ligated into an appropriate expression vector. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential test sequences.The synthesis of degenerate oligonucleotides is well known in the art(see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.(1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed.AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu.Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

[0196] As used herein, “variegated” refers to the fact that a populationof peptides is characterized by having a peptide sequence which differfrom one member of the library to the next. For example, in a givenpeptide library of n amino acids in length, the total number ofdifferent peptide sequences in the library is given by the product of{ν₁×ν₂× . . . ν_(n−1)×ν_(n)} where each □_(n) represents the numberdifferent amino acid residues occurring at position n of the peptide. Ina preferred embodiment of the present invention, the peptide displaycollectively produces a peptide library including at least 96 to 10⁷different peptides, so that diverse peptides may be simultaneouslyassayed for the ability to interact with the target protein.

[0197] In one embodiment, the test peptide library is derived to expressa combinatorial library of peptides which are not based on any knownsequence, nor derived from cDNA. That is, the sequences of the libraryare largely, if not entirely, random. It will be evident that thepeptides of the library may range in size from dipeptides to largeproteins.

[0198] In another embodiment, the peptide library is derived to expressa combinatorial library of peptides which are based at least in part ona known polypeptide sequence or a portion thereof (though preferably nota CDNA library). That is, the sequences of the library is semi-random,being derived by combinatorial mutagenesis of a known sequence(s). See,for example, Ladner et al. PCT publication WO 90/02909; Garrard et al.,PCT publication WO 92/09690; Marks et al. (1992) J Biol. Chem.267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461. Accordingly, polypeptide(s) which are known ligands for atarget protein can be mutagenized by standard techniques to derive avariegated library of polypeptide sequences which can further bescreened for agonists and/or antagonists. The purpose of screening suchcombinatorial peptide libraries is to generate, for example, homologs ofknown polypeptides which can act as either agonists or antagonists, oralternatively, possess novel activities all together. To illustrate, aligand can be engineered by the present method to provide more efficientbinding or specificity to a cognate receptor, yet still retain at leasta portion of an activity associated with wild-type ligand. Thus,combinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein. Likewise,homologs can be generated by the present approach to act as antagonists,in that they are able to mimic, for example, binding to the target, yetnot induce any biological response, thereby inhibiting the action ofauthentic ligand.

[0199] In preferred embodiments, the combinatorial polypeptides are inthe range of 3-100 amino acids in length, more preferably at least 5-50,and even more preferably at least 10, 13, 15, 20 or 25 amino acidresidues in length. Preferably, the polypeptides of the library are ofuniform length. It will be understood that the length of thecombinatorial peptide does not reflect any extraneous sequences whichmay be present in order to facilitate expression, e.g., such as signalsequences or invariant portions of a fusion protein.

[0200] In preferred embodiments, the test peptide is flanked by cysteineresidues in order to provide a constrained environment. For example, thetest peptide may be represented in the general formulaCys-(Xaa)₃₋₂₃-Cys.

[0201] The harnessing of biological systems for the generation ofpeptide diversity is now a well established technique which can beexploited to generate the peptide libraries of the subject method. Thesource of diversity is the combinatorial chemical synthesis of mixturesof oligonucleotides. Oligonucleotide synthesis is a well-characterizedchemistry that allows tight control of the composition of the mixturescreated. Degenerate DNA sequences produced are subsequently placed intoan appropriate genetic context for expression as peptides.

[0202] There are two principal ways in which to prepare the requireddegenerate mixture. In one method, the DNAs are synthesized a base at atime. When variation is desired at a base position dictated by thegenetic code a suitable mixture of nucleotides is reacted with thenascent DNA, rather than the pure nucleotide reagent of conventionalpolynucleotide synthesis. The second method provides more exact controlover the amino acid variation. First, trinucleotide reagents areprepared, each trinucleotide being a codon of one (and only one) of theamino acids to be featured in the peptide library. When a particularvariable residue is to be synthesized, a mixture is made of theappropriate trinucleotides and reacted with the nascent DNA. Once thenecessary “degenerate” DNA is complete, it must be joined with the DNAsequences necessary to assure the expression of the peptide, asdiscussed in more detail below, and the complete DNA construct must beintroduced into the cell.

[0203] Whatever the method may be for generating diversity at the codonlevel, chemical synthesis of a degenerate gene sequence can be carriedout in an automatic DNA synthesizer, and the synthetic genes can then beligated into an appropriate gene for expression. The purpose of adegenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential test peptide sequences.The synthesis of degenerate oligonucleotides is well known in the art(see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.(1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed.AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu.Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

[0204] IV. Exemplary Uses

[0205] Because of the flexibility of the system, the subject method canbe used in a broad range of applications, including for the selection ofpeptides having effects on proliferation, differentiation, cell death,cell migration, etc. In preferred embodiments, the target used in thedisplay mode is an extracellular component of a cell. However, it willbe appreciated that the target for subject method can be anintracellular component and, during the secretion mode, the system canbe augmented with agents which promote the cellular uptake of the testpeptides.

[0206] In an illustrative embodiment, the subject method is utilized toidentify peptides which have antiproliferative activity with respect toone or more types of cells. For instance, in the display mode, thepeptide library can be panned with the target cells for which anantiproliferative is desired in order to enrich for peptides which bindto that cell. At that stage, the peptide library can also be pannedagainst one or more control cell lines in order to remove peptides whichbind the control cells. In this manner, the peptide library which isthen tested in the secretion mode can be enriched for peptides whichselectively bind target cell (relative to the control cells). Thus, forexample, the display mode can produce a peptide library enriched forpeptides which preferentially bind tumor cells relative to normal cells,which preferentially bind p53− cells relative to p53+ cells, whichpreferentially bind hair follicle cells relative to other epithelialcells, or any other differential binding characteristic.

[0207] In the secretion mode, the peptides are tested forantiproliferative activity against the target cell using any of a numberof techniques known in the art. For instance, BrdU or other nucleotideuptake can be measured as an indicator of proliferation. As above, thesecretion mode can include negative controls in order to select forpeptides with specific antiproliferative activity.

[0208] In similar fashion, peptides can be isolated from the librarybased on their ability to induce apoptosis or cell lysis, e.g., in acell selective manner.

[0209] In yet another embodiment, the subject method can be used toidentify peptides with angiogenic or antiangiogenic activity. Forinstance, as illustrated in FIG. 6, the peptide library can be enrichedfor peptides that bind to endothelial cells but which do not bind tofibroblasts. The resulting sub-library can be screened for peptideswhich inhibit capillary endothelial cell proliferation and/orendothelial cell migration. Peptides scoring positive for one or both ofthese activities can also be tested for activity against other celltypes, such as smooth muscle cells or fibroblasts, in order to selectpeptides active only against endothelial cells.

[0210] In still another embodiment, the subject method can be used toidentify anti-infective peptides, e.g., which are active as anti-fungalor antibacterial agents.

[0211] In one embodiment, the assay of the present invention can be usedfor identifying effectors of a receptor protein or complex thereof. Ingeneral, the assay is characterized by the use of a test cell whichincludes a target receptor or ion channel protein whose signaltransduction activity can be modulated by interaction with anextracellular signal, the transduction activity being able to generate adetectable signal.

[0212] In general, such embodiments of the subject assay arecharacterized by the use of a mixture of cells expressing a targetreceptor protein or ion channel capable of transducing a detectablesignal in the reagent cell. The receptor/channel protein can be eitherendogenous or heterologous. In combination with the disclosed detectionmeans, a culture of the instant reagent cells will provide means fordetecting agonists or antagonists of receptor function.

[0213] The ability of particular peptides to modulate a signaltransduction activity of the target receptor or channel can be scoredfor by detecting up or down-regulation of the detection signal. Forexample, second messenger generation (e.g. GTPase activity, phospholipidhydrolysis, or protein phosphorylation patterns as examples) can bemeasured directly. Alternatively, the use of an indicator gene canprovide a convenient readout. In other embodiments a detection meansconsists of an indicator gene. In any event, a statistically significantchange in the detection signal can be used to facilitate identificationof compounds which modulate receptor or ion channel activities.

[0214] By this method, peptides which induce a signal pathway from aparticular receptor or channel can be identified. If a test peptide doesnot appear to induce the activity of the receptor/channel protein, theassay may be repeated as described above, and modified by theintroduction of a step in which the reagent cell is first contacted witha known activator of the target receptor/channel to induce signaltransduction, and the test peptide can be assayed for its ability toinhibit the activated receptor/channel, e.g., to identify antagonists.In yet other embodiments, peptides can be screened for those whichpotentiate the response to a known activator of the receptor.

[0215] With respect to the receptor or ion channel, it may beendogenously expressed by the host cell, or it may be expressed from aheterologous gene that has been introduced into the cell. Methods forintroducing heterologous DNA into eukaryotic cells are of course wellknown in the art and any such method may be used. In addition, DNAencoding various receptor proteins is known to those of skill in the artor it may be cloned by any method known to those of skill in the art. Incertain embodiments, such as when an exogenous receptor is expressed, itmay be desirable to inactivate, such as by deletion, a homologousreceptor present in the cell.

[0216] In particular, the assays can be used to test functionalligand-receptor or ligand-ion channel interactions for cellsurface-localized receptors and channels. As described in more detailbelow, the subject assay can be used to identify effectors of, forexample, G protein-coupled receptors, receptor tyrosine kinases,cytokine receptors, and ion channels. In certain embodiments the methoddescribed herein is used for identifying ligands for “orphan receptors”for which no ligand is known.

[0217] In preferred embodiments, the receptor is a cell surfacereceptor, such as: a receptor tyrosine kinase, e.g., an EPH receptor; anion channel; a cytokine receptor; an multisubunit immune recognitionreceptor, a chemokine receptor; a growth factor receptor, or a G-proteincoupled receptor, such as a chemoattracttractant peptide receptor, aneuropeptide receptor, a light receptor, a neurotransmitter receptor, ora polypeptide hormone receptor.

[0218] Preferred G protein coupled receptors include α1A-adrenergicreceptor, α1B-adrenergic receptor, α2-adrenergic receptor,α2B-adrenergic receptor, β1-adrenergic receptor, β2-adrenergic receptor,β3-adrenergic receptor, m1 acetylcholine receptor (AChR), m2 AChR, m3AChR, m4 AChR, m5 AChR, D1 dopamine receptor, D2 dopamine receptor, D3dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, A1adenosine receptor, A2b adenosine receptor, 5-HT1a receptor, 5-HT1breceptor, 5HT1-like receptor, 5-HT1d receptor, 5HT1d-like receptor,5HT1d beta receptor, substance K (neurokinin A) receptor, fMLP receptor,fMLP-like receptor, angiotensin II type 1 receptor, endothelin ETAreceptor, endothelin ETB receptor, thrombin receptor, growthhormone-releasing hormone (GHRH) receptor, vasoactive intestinal peptidereceptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3,cannabinoid receptor, follicle stimulating hormone (FSH) receptor,leutropin (LH/HCG) receptor, thyroid stimulating hormone (TSH) receptor,thromboxane A2 receptor, platelet-activating factor (PAF) receptor, C5aanaphylatoxin receptor, Interleukin 8 (IL-8) IL-8RA, IL-8RB, DeltaOpioid receptor, Kappa Opioid receptor, mip-1/RANTES receptor,Rhodopsin, Red opsin, Green opsin, Blue opsin, metabotropic glutamatemGluR1-6, histamine H2 receptor, ATP receptor, neuropeptide Y receptor,amyloid protein precursor receptor, insulin-like growth factor IIreceptor, bradykinin receptor, gonadotropin-releasing hormone receptor,cholecystokinin receptor, melanocyte stimulating hormone receptorreceptor, antidiuretic hormone receptor, glucagon receptor, andadrenocorticotropic hormone II receptor.

[0219] Preferred EPH receptors inlcude eph, elk, eck, sek, mek4, hek,hek2, eek, erk, tyrol, tyro4, tyro5, tyro6, tyroll, cek4, cek5, cek6,cek7, cek8, cek9, cek10, bsk, rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2,pagliaccio, htk, erk and nuk receptors.

[0220] A. Cytokine Receptors

[0221] In one embodiment the target receptor is a cytokine receptor.Cytokines are a family of soluble mediators of cell-to-cellcommunication that includes interleukins, interferons, andcolony-stimulating factors. The characteristic features of cytokines liein their functional redundancy and pleiotropy. Most of the cytokinereceptors that constitute distinct superfamilies do not possessintrinsic protein tyrosine kinase domains, yet receptor stimulationusually invokes rapid tyrosine phosphorylation of intracellularproteins, including the receptors themselves. Many members of thecytokine receptor superfamily acitvate the Jak protein tyrosine kinasefamily, with resultant phosphorylation of the STAT transcriptionalactivator factors. IL-2, IL-7, IL-2 and Interferon □ have all been shownto activate Jak kinases (Frank et al (1995) Proc Natl Acad Sci USA92:7779-7783); Scharfe et al. (1995) Blood 86:2077-2085); (Bacon et al.(1995) Proc Natl Acad Sci USA 92:7307-7311); and (Sakatsume et al (1995)J Biol Chem 270:17528-17534). Events downstream of Jak phosphorylationhave also been elucidated. For example, exposure of T lymphocytes toIL-2 has been shown to lead to the phosphorylation of signal transducersand activators of transcription (STAT) proteins STAT1□, STAT2□, andSTAT3, as well as of two STAT-related proteins, p94 and p95. The STATproteins were found to translocate to the nucleus and to bind to aspecific DNA sequence, thus suggesting a mechanism by which IL-2 mayactivate speicfic genes involved in immune cell function (Frank et al.supra). Jak3 is associated with the gamma chain of the IL-2, IL-4, andIL-7 cytokine receptors (Fujii et al. (1995) Proc Natl Acad Sci92:5482-5486) and (Musso et al (1995) J Exp Med. 181:1425-1431). The Jakkinases have also been shown to be activated by numerous ligands thatsignal via cytokine receptors such as, growth hormone and erythropoietinand IL-6 (Kishimoto (1994) Stem cells Suppl 12:37-44).

[0222] Detection means which may be scored for in the present assay, inaddition to direct detection of second messangers, such as by changes inphosphorylation, includes reporter constructs or indicator genes whichinclude transcriptional regulatory elements responsive to the STATproteins. Described infra.

[0223] B Multisubunit Immune Recognition Receptor (MIRR).

[0224] In another embodiment the receptor is a multisubunit receptor.Receptors can be comprised of multiple proteins referred to as subunits,one category of which is referred to as a multisubunit receptor is amultisubunit immune recognition receptor (MIRR). MIRRs include receptorshaving multiple noncovalently associated subunits and are capable ofinteracting with src-family tyrosine kinases. MIRRs can include, but arenot limited to, B cell antigen receptors, T cell antigen receptors, Fcreceptors and CD22. One example of an MIRR is an antigen receptor on thesurface of a B cell. To further illustrate, the MIRR on the surface of aB cell comprises membrane-bound immunoglobulin (mIg) associated with thesubunits Ig-α and Ig-β or Ig-γ, which forms a complex capable ofregulating B cell function when bound by antigen. An antigen receptorcan be functionally linked to an amplifier molecule in a manner suchthat the amplifier molecule is capable of regulating gene transcription.

[0225] Src-family tyrosine kinases are enzymes capable ofphosphorylating tyrosine residues of a target molecule. Typically, asrc-family tyrosine kinase contains one or more binding domains and akinase domain. A binding domain of a src-family tyrosine kinase iscapable of binding to a target molecule and a kinase domain is capableof phosphorylating a target molecule bound to the kinase. Members of thesrc family of tyrosine kinases are characterized by an N-terminal uniqueregion followed by three regions that contain different degrees ofhomology among all the members of the family. These three regions arereferred to as src homology region 1 (SH1), src homology region 2 (SH2)and src homology region 3 (SH3). Both the SH2 and SH3 domains arebelieved to have protein association functions important for theformation of signal transduction complexes. The amino acid sequence ofan N-terminal unique region, varies between each src-family tyrosinekinase. An N-terminal unique region can be at least about the first 40amino acid residues of the N-terminal of a src-family tyrosine kinase.

[0226] Syk-family kinases are enzymes capable of phosphorylatingtyrosine residues of a target molecule. Typically, a syk-family kinasecontains one or more binding domains and a kinase domain. A bindingdomain of a syk-family tyrosine kinase is capable of binding to a targetmolecule and a kinase domain is capable of phosphorylating a targetmolecule bound to the kinase. Members of the syk- family of tyrosinekinases are characterized by two SH2 domains for protein associationfunction and a tyrosine kinase domain.

[0227] A primary target molecule is capable of further extending asignal transduction pathway by modifying a second messenger molecule.Primary target molecules can include, but are not limited to,phosphatidylinositol 3-kinase (PI-3K), P21^(ras)GAPase-activatingprotein and associated P190 and P62 protein, phospholipases such asPLCγ1 and PLCγ2, MAP kinase, Shc and VAV. A primary target molecule iscapable of producing second messenger molecule which is capable offurther amplifying a transduced signal. Second messenger moleculesinclude, but are not limited to diacylglycerol and inositol1,4,5-triphosphate (IP3). Second messenger molecules are capable ofinitiating physiological events which can lead to alterations in genetranscription. For example, production of IP3 can result in release ofintracellular calcium, which can then lead to activation of calmodulinkinase II, which can then lead to serine phosphorylation of a DNAbinding protein referred to as ets-1 proto-onco-protein. Diacylglycerolis capable of activating the signal transduction protein, protein kinaseC which affects the activity of the AP1 DNA binding protein complex.Signal transduction pathways can lead to transcriptional activation ofgenes such as c-fos, egr-1, and c-myc.

[0228] Shc can be thought of as an adaptor molecule. An adaptor moleculecomprises a protein that enables two other proteins to form a complex(e.g., a three molecule complex). Shc protein enables a complex to formwhich includes Grb2 and SOS. Shc comprises an SH2 domain that is capableof associating with the SH2 domain of Grb2.

[0229] Molecules of a signal transduction pathway can associate with oneanother using recognition sequences. Recognition sequences enablespecific binding between two molecules. Recognition sequences can varydepending upon the structure of the molecules that are associating withone another. A molecule can have one or more recognition sequences, andas such can associate with one or more different molecules.

[0230] Signal transduction pathways for MIRR complexes are capable ofregulating the biological functions of a cell. Such functions caninclude, but are not limited to the ability of a cell to grow, todifferentiate and to secrete cellular products. MIRR-induced signaltransduction pathways can regulate the biological functions of specifictypes of cells involved in particular responses by an animal, such asimmune responses, inflammatory responses and allergic responses. Cellsinvolved in an immune response can include, for example, B cells, Tcells, macrophages, dendritic cells, natural killer cells and plasmacells. Cells involved in inflammatory responses can include, forexample, basophils, mast cells, cosinophils, neutrophils andmacrophages. Cells involved in allergic responses can include, forexample mast cells, basophils, B cells, T cells and macrophages.

[0231] In exemplary embodiments of the subject assay, the detectionsignal is a second messengers, such as a phosphorylated src-likeprotein, includes reporter constructs or in dicator genes which includetranscriptional regulatory elements such as serum response element(SRE), 12-O-tetradecanoyl-phorbol-13-acetate response element, cyclicAMP response element, c-fos promoter, or a CREB-responsive element.

[0232] C. Receptor tyrosine kinases.

[0233] In still another embodiment, the target receptor is a receptortyrosine kinase. The receptor tyrosine kinases can be divided into fivesubgroups on the basis of structural similarities in their extracellulardomains and the organization of the tyrosine kinase catalytic region intheir cytoplasmic domains. Sub-groups I (epidermal growth factor (EGF)receptor-like), II (insulin receptor-like) and the eph/eck familycontain cysteine-rich sequences (Hirai et al., (1987) Science238:1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol.10:6316-6324). The functional domains of the kinase region of thesethree classes of receptor tyrosine kinases are encoded as a contiguoussequence ( Hanks et al. (1988) Science 241:42-52). Subgroups III(platelet-derived growth factor (PDGF) receptor-like) and IV (thefibro-blast growth factor (FGF) receptors) are characterized as havingimmunoglobulin (Ig)-like folds in their extracellular domains, as wellas having their kinase domains divided in two parts by a variablestretch of unrelated amino acids (Yanden and Ullrich (1988) supra andHanks et al. (1988) supra).

[0234] The family with by far the largest number of known members is theEPH family. Since the description of the prototype, the EPH receptor(Hirai et al. (1987) Science 238:1717-1720), sequences have beenreported for at least ten members of this family, not countingapparently orthologous receptors found in more than one species.Additional partial sequences, and the rate at which new members arestill being reported, suggest the family is even larger (Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech Dev, 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J Neurosci Res 37:129-143; and references in Tuzi andGullick (1994) Br J Cancer 69:417-421). Remarkably, despite the largenumber of members in the EPH family, all of these molecules wereidentified as orphan receptors without known ligands.

[0235] The expression patterns determined for some of the EPH familyreceptors have implied important roles for these molecules in earlyvertebrate development., In particular, the timing and pattern ofexpression of sek, mek4 and some of the other receptors, during thephase of gastrulation and early organogenesis has suggested functionsfor these receptors in the important cellular interactions involved inpatterning the embryo at this stage (Gilardi-Hebenstreit et al. (1992)Oncogene 7:2499-2506; Nieto et al. (1992) Development 116:1137-1150;Henkemeyer et al., supra; Ruiz et al., supra; and Xu et al., supra).Sek, for example, shows a notable early expression in the two areas ofthe mouse embryo that show obvious segmentation, namely the somites inthe mesoderm and the rhombomeres of the hindbrain; hence the name sek,for segmentally expressed kinase (Gilardi-Hebenstreit et al., supra;Nieto et al., supra). As in Drosophila, these segmental structures ofthe mammalian embryo are implicated as important elements inestablishing the body plan. The observation that Sek expression precedesthe appearance of morphological segmentation suggests a role for sek informing these segmental structures, or in determining segment-specificcell properties such as lineage compartmentation (Nieto et al., supra).Moreover, EPH receptors have been implicated, by their pattern ofexpression, in the development and maintenance of nearly every tissue inthe embryonic and adult body. For instance, EPH receptors have beendetected throughout the nervous system, the testes, the cartilaginousmodel of the skeleton, tooth primordia, the infundibular component ofthe pituitary, various epithelia tissues, lung, pancreas, liver andkidney tissues. Observations such as this have been indicative ofimportant and unique roles for EPH family kinases in development andphysiology, but further progress in understanding their action has beenseverely limited by the lack of information on their ligands.

[0236] As used herein, the terms “EPH receptor” or “EPH-type receptor”refer to a class of receptor tyrosine kinases, comprising at leasteleven paralogous genes, though many more orthologs exist within thisclass, e.g. homologs from different species. EPH receptors, in general,are a discrete group of receptors related by homology and easilyreconizable, e.g., they are typically characterized by an extracellulardomain containing a characteristic spacing of cysteine residues near theN-terminus and two fibronectin type III repeats (Hirai et al. (1987)Science 238:1717-1720; Lindberg et al. (1990) Mol Cell Biol10:6316-6324; Chan et al. (1991) Oncogene 6:1057-1061; Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J Neurosci Res 37:129-143; and references in Tuzi andGullick (1994) Br J Cancer 69:417-421). Exemplary EPH receptors includethe eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyro1, tyro4, tyro5,tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk, rtk1,rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio, htk, erk and nukreceptors. The term “EPH receptor” refers to the membrane form of thereceptor protein, as well as soluble extracellular fragments whichretain the ability to bind the ligand of the present invention.

[0237] In exemplary embodiments, the detection signal is provided bydetecting phosphorylation of intracellular proteins, e.g., MEKKs, MEKs,or Map kinases, or by the use of reporter constructs or indicator geneswhich include transcriptional regulatory elements responsive to c-fosand/or c-jun. Described infra.

[0238] D. G Protein-Coupled Receptors.

[0239] One family of signal transduction cascades found in eukaryoticcells utilizes heterotrinieric “G proteins.” Many different G proteinsare known to interact with receptors. G protein signaling systemsinclude three components: the receptor itself, a GTP-binding protein (Gprotein), and an intracellular target protein.

[0240] The cell membrane acts as a switchboard. Messages arrivingthrough different receptors can produce a single effect if the receptorsact on the same type of G protein. On the other hand, signals activatinga single receptor can produce more than one effect if the receptor actson different kinds of G proteins, or if the G proteins can act ondifferent effectors.

[0241] In their resting state, the G proteins, which consist of alpha(α), beta (β) and gamma (γ) subunits, are complexed with the nucleotideguanosine diphosphate (GDP) and are in contact with receptors. When ahormone or other first messenger binds to receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the α subunit to release GDP, and the more abundant hucleotideguanosine triphosphate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the α subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active “second messenger,” which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the Gβγ complex.

[0242] Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the asubunit, several different β and γ structures have been reported. Thereare, additionally, several different G protein-dependent effectors.

[0243] Most G protein-coupled receptors are comprised of a singleprotein chain that is threaded through the plasma membrane seven times.Such receptors are often referred to as seven-transmembrane receptors(STRs). More than a hundred different STRs have been found, includingmany distinct receptors that bind the same ligand, and there are likelymany more STRs awaiting discovery.

[0244] In addition, STRs have been identified for which the naturalligands are unknown; these receptors are termed “orphan” Gprotein-coupled receptors, as described above. Examples includereceptors cloned by Neote et al. (1993) Cell 72, 415; Kouba et al. FEBSLett. (1993) 321, 173; Birkenbach et al.(1993) J Virol. 67, 2209.

[0245] The “exogenous receptors” of the present invention may be any Gprotein-coupled receptor which is exogenous to the cell which is to begenetically engineered for the purpose of the present invention. Thisreceptor may be a plant or animal cell receptor. Screening for bindingto plant cell receptors may be useful in the development of, e.g.,herbicides. In the case of an animal receptor, it may be of invertebrateor vertebrate origin. If an invertebrate receptor, an insect receptor ispreferred, and would facilitate development of insecticides. Thereceptor may also be a vertebrate, more preferably a mammalian, stillmore preferably a human, receptor. The exogenous receptor is alsopreferably a seven transmembrane segment receptor.

[0246] Known ligands for G protein coupled receptors include: purinesand nucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin andthe like; biogenic amines (and related natural ligands), such as5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, adrenaline,adrenaline., histamine, noradrenaline, noradrenaline, noradrenaline.,tyramine/octopamine and other related compounds; peptides such asadrenocorticotrophic hormone (acth), melanocyte stimulating hormone(msh), melanocortins, neurotensin (nt), bombesin and related peptides,endothelins, cholecystokinin, gastrin, neurokinin b (nk3), invertebratetachykinin-like peptides, substance k (nk2), substance p (nk1),neuropeptide y (npy), thyrotropin releasing-factor (trf), bradykinin,angiotensin ii, beta-endorphin, c5a anaphalatoxin, calcitonin,chemokines (also called intercrines), corticotrophic releasing factor(crf), dynorphin, endorphin, fmlp and other formylated peptides,follitropin (fsh), fungal mating pheremones, galanin, gastric inhibitorypolypeptide receptor (gip), glucagon-like peptides (glps), glucagon,gonadotropin releasing hormone (gnrh), growth hormone releasinghormone(ghrh), insect diuretic hormone, interleukin-8, leutropin(1h/hcg), metenkephalin, opioid peptides, oxytocin, parathyroid hormone(pth) and pthrp, pituitary adenylyl cyclase activiating peptide (pacap),secretin, somatostatin, thrombin, thyrotropin (tsh), vasoactiveintestinal peptide (vip), vasopressin, vasotocin; eicosanoids such asip-prostacyclin, pg-prostaglandins, tx-thromboxanes; retinal basedcompounds such as vertebrate 11-cis retinal, invertebrate 11-cis retinaland other related compounds; lipids and lipid-based compounds such ascannabinoids, anandamide, lysophosphatidic acid, platelet activatingfactor, leukotrienes and the like; excitatory amino acids and ions suchas calcium ions and glutamate.

[0247] Suitable examples of G-protein coupled receptors include, but arenot limited to, dopaminergic, muscarinic cholinergic, a-adrenergic,b-adrenergic, opioid (including delta and mu), cannabinoid,serotoninergic, and GABAergic receptors. Preferred receptors include the5HT family of receptors, dopamine receptors, C5a receptor and FPRL-1receptor, cyclo-histidyl-proline-diketoplperazine receptors, melanocytestimulating hormone release inhibiting factor receptor, and receptorsfor neurotensin, thyrotropin releasing hormone, calcitonin,cholecytokinin-A, neurokinin-2, histamine-3, cannabinoid, melanocortin,or adrenomodulin, neuropeptide-Y1 or galanin. Other suitable receptorsare listed in the art. The term “receptor,” as used herein, encompassesboth naturally occurring and mutant receptors.

[0248] Many of these G protein-coupled receptors, like the yeast a- and□-factor receptors, contain seven hydrophobic amino acid-rich regionswhich are assumed to lie within the plasma membrane. Specific human Gprotein-coupled STRs for which genes have been isolated and for whichexpression vectors could be constructed include those listed herein andothers known in the art. Thus, the gene would be operably linked to apromoter functional in the cell to be engineered and to a signalsequence that also functions in the cell. For example in the case ofyeast, suitable promoters include Ste2, Ste3 and gal10. Suitable signalsequences include those of Ste2, Ste3 and of other genes which encodeproteins secreted by yeast cells. Preferably, when a yeast cell is used,the codons of the gene would be optimized for expression in yeast. SeeHoekema et al.,(1987)Mol. Cell. Biol., 7:2914-24; Sharp, et al.,(1986)14:5125-43.

[0249] The homology of STRs is discussed in Dohlman et al., Ann. Rev.Biochem., (1991) 60:653-88. When STRs are compared, a distinct spatialpattern of homology is discernible. The transmembrane domains are oftenthe most similar, whereas the N- and C-terminal regions, and thecytoplasmic loop connecting transmembrane segments V and VI are moredivergent.

[0250] The functional significance of different STR regions has beenstudied by introducing point mutations (both substitutions anddeletions) and by constructing chimeras of different but related STRs.Synthetic peptides corresponding to individual segments have also beentested for activity. Affinity labeling has been used to identify ligandbinding sites.

[0251] It is conceivable that when the host cell is a yeast cell, aforeign receptor will fail to functionally integrate into the yeastmembrane, and there interact with the endogenous yeast G protein. Morelikely, either the receptor will need to be modified (e.g., by replacingits V-VI loop with that of the yeast STE2 or STE3 receptor), or acompatible G protein should be provided.

[0252] If the wild-type exogenous G protein-coupled receptor cannot bemade functional in yeast, it may be mutated for this purpose. Acomparison would be made of the amino acid sequences of the exogenousreceptor and of the yeast receptors, and regions of high and lowhomology identified. Trial mutations would then be made to distinguishregions involved in ligand or G protein binding, from those necessaryfor functional integration in the membrane. The exogenous receptor wouldthen be mutated in the latter region to more closely resemble the yeastreceptor, until functional integration was achieved. If this wereinsufficient to achieve functionality, mutations would next be made inthe regions involved in G protein binding. Mutations would be made inregions involved in ligand binding only as a last resort, and then aneffort would be made to preserve ligand binding by making conservativesubstitutions whenever possible.

[0253] Preferably, the yeast genome is modified so that it is unable toproduce the yeast receptors which are homologous to the exogenousreceptors in functional form. Otherwise, a positive assay score mightreflect the ability of a peptide to activate the endogenous Gprotein-coupled receptor, and not the receptor of interest.

[0254] (i). Chemoattractant receptors

[0255] The N-formyl peptide receptor is a classic example of a calciummobilizing G protein-coupled receptor expressed by neutrophils and otherphagocytic cells of the mammalian immune system (Snyderman et al. (1988)In Inflammation: Basic Principles and Clinical Correlates, pp. 309-323).N-formyl peptides of bacterial origin bind to the receptor and engage acomplex activation program that results in directed cell movement,release of inflammatory granule contents, and activation of a latentNADPH oxidase which is important for the production of metabolites ofmolecular oxygen. This pathway initiated by receptor-ligand interactionis critical in host protection from pyogenic infections. Similar signaltransduction occurs in response to the inflammatory peptides C5a andIL-8.

[0256] Two other formyl peptide receptor like (FPRL) genes have beencloned based on their ability to hybridize to a fragment of the NFPRcDNA coding sequence. These have been named FPRL1 (Murphy et al. (1992)J. Biol Chem. 267:7637-7643) and FPRL2 (Ye et al. (1992) Biochem BiophysRes. Comm. 184:582-589). FPRL2 was found to mediate calcium mobilizationin mouse fibroblasts transfected with the gene and exposed to formylpeptide. In contrast, although FPRL1 was found to be 69% identical inamino acid sequence to NFPR, it did not bind prototype N-formyl peptidesligands when expressed in heterologous cell types. This lead to thehypothesis of the existence of an as yet unidentified ligand for theFPRL1 orphan receptor (Murphy et al. supra).

[0257] (ii.) G proteins

[0258] In the case of an exogenous G-protein coupled receptor, the yeastcell must be able to produce a G protein which is activated by theexogenous receptor, and which can in turn activate the yeasteffector(s). The art suggests that the endogenous yeast Gα subunit(e.g., GPA) will be often be sufficiently homologous to the “cognate” Gαsubunit which is natively associated with the exogenous receptor forcoupling to occur. More likely, it will be necessary to geneticallyengineer the yeast cell to produce a foreign Gα subunit which canproperly interact with the exogenous receptor. For example, the Gαsubunit of the yeast G protein may be replaced by the Gα subunitnatively associated with the exogenous receptor.

[0259] Dietzel and Kurjan, (1987) Cell, 50:1001) demonstrated that ratGas functionally coupled to the yeast Gβγ complex. However, rat Gαi2complemented only when substantially overexpressed, while Gα0 did notcomplement at all. Kang, et al., Mol. Cell. Biol., (1990)10:2582).Consequently, with some foreign Gα subunits, it is not feasible tosimply replace the yeast Gα.

[0260] If the exogenous G protein coupled receptor is not adequatelycoupled to yeast Gβγ by the Gα subunit natively associated with thereceptor, the Gα subunit may be modified to improve coupling. Thesemodifications often will take the form of mutations which increase theresemblance of the Gα subunit to the yeast Gα while decreasing itsresemblance to the receptor-associated Gα. For example, a residue may bechanged so as to become identical to the corresponding yeast Gα residue,or to at least belong to the same exchange group of that residue. Aftermodification, the modified Gα subunit might or might not be“substantially homologous” to the foreign and/or the yeast Gα subunit.

[0261] The modifications are preferably concentrated in regions of theGα which are likely to be involved in Gβγ binding. In some embodiments,the modifications will take the form of replacing one or more segmentsof the receptor-associated Gα with the corresponding yeast Gαsegment(s), thereby forming a chimeric Gα subunit. (For the purpose ofthe appended claims, the term “segment” refers to three or moreconsecutive amino acids.) In other embodiments, point mutations may besufficient.

[0262] This chirneric Gα subunit will interact with the exogenousreceptor and the yeast Gβγ complex, thereby permitting signaltransduction. While use of the endogenous yeast Gβγ is preferred, if aforeign or chimeric Gβγ is capable of transducing the signal to theyeast effector, it may be used instead.

[0263] V. Pharmaceutical Preparations of Identifled Agents

[0264] After identifying certain test peptides in the subject assay,e.g. as potential surrogate ligands, or receptor antagonists, thepractitioner of the subject assay will continue to test the efficacy andspecificity of the selected peptides both in vitro and in vivo. Whetherfor subsequent in vivo testing, or for administration to an animal as anapproved drug, peptides identified in the subject assay, orpeptidomimetics thereof, can be formulated in pharmaceuticalpreparations for in vivo administration to an animal, preferably ahuman.

[0265] The peptides selected in the subject assay, or a pharmaceuticallyacceptable salt thereof, may accordingly be formulated foradministration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thecompound, its use in the pharmaceutical preparation of the invention iscontemplated. Suitable vehicles and their formulation inclusive of otherproteins are described, for example, in the book Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences. MackPublishing Company, Easton, Pa., USA 1985). These vehicles includeinjectable “deposit formulations”. Based on the above, suchpharmaceutical formulations include, although not exclusively, solutionsor freeze-dried powders of the compound in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered media at a suitable pH and isosmotic with physiological fluids.In preferred embodiment, the peptide can be disposed in a sterilepreparation for topical and/or systemic administration. In the case offreeze-dried preparations, supporting excipients such as, but notexclusively, mannitol or glycine may be used and appropriate bufferedsolutions of the desired volume will be provided so as to obtainadequate isotonic buffered solutions of the desired pH. Similarsolutions may also be used for the pharmaceutical compositions ofcompounds in isotonic solutions of the desired volume and include, butnot exclusively, the use of buffered saline solutions with phosphate orcitrate at suitable concentrations so as to obtain at all times isotonicpharmaceutical preparations of the desired pH, (for example, neutralpH).

Exemplification

[0266] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention.

[0267] As shown in FIG. 1, pAM6 M13/COS peptide expression plasmid isdesigned to have distinct functions in prokaryotic and eukaryotic cells.In prokaryotic cells the plasmid functions in the display mode, whichdirects the display of encoded peptides on the surface of the M13 phagewithin which it is packaged. The peptides are displayed as fusions withthe major M13 capsid protein pVIII. The depicted elements which enablethis mode are as follows: lac, the prokaryotic promoter; preVIII signal,the periplasmic targeting signal sequence from M13 geneIII (the topvertical arrows which follow indicate cleavage sites for the signalpeptidase); random peptide, the random or specific coding sequence ofthe peptide to be fused to an M13 coat protein; M13 pVII, the codingsequence of the pVIII coat protein of M13 (the STOP above it representsthe stop codon and prokaryotic transcriptional terminator); ApR, the13-lactamase gene which allows ampicillin selection for cells carryingthe plasmid; pUC ori, replication origin which directs high copy numberreplication of the plasmid within E. coli; M13(−) ori, replicationorigin and packaging signal which, in the presence of M13 helper phage,allows generation of single stranded DNA, and directs its packaging intophage particles.

[0268] In eukaryotic cells the plasmid functions in the secretion mode,which results in the secretion of the encoded peptide, apart from othersequences, into the extracellular media. The depicted elements whichenable this mode are as follows: CMV enh/prom, the eukaryotic promoter;IgH secr.s., the immunoglobulin heavy chain signal sequence whichdirects extracellular secretion of proteins; random peptide, the randomor specific coding sequence of the peptide to be secreted; SV40 polyA,RNA polyadenylation signal; globin splice donor/IgH splice acceptorpairs, direct processing of the RNA to a mature mRNA that is devoid ofthe unwanted intervening prokaryotic sequences; SV40 ori, origin ofreplication which results in high copy number replication of the plasmidin COS cells.

[0269] Other exemplary plasmids, pAM7 and pAM9 M13/COS peptideexpression plasmids, are shown in FIG. 2. The difference between pAM7and pAM9 is the M13 coat protein to which the peptide is fused in thedisplay mode: pAM7 utilizes a pVIII fusion and pAM9 utilizes a pIIIfusion, as indicated by M13 pVIII or pIII.

[0270] This plasmid design is identical to pAM6 (see FIG. 1) with theexception of the following modifications: The CMV enh/prom and lacpromoters have been separated and placed adjacent to their respectiveeukaryotic IgH signal s and prokaryotic E.coli signal s signalsequences. The first pair of splice signals now functions to remove thelac promoter and E.coli signal sequence from the mRNA, as opposed tojust the signal sequence in pAM6. In addition, the E.coli signal ssequence was created from concensus signal sequences, and into it'scoding sequence the IgH splice acceptor sequence was silentlyengineered, which eliminates the addition of extra residues onto theamino terminus of the random peptide.

[0271]FIG. 3 illustrates yet another plasmid of the invention, the pAM8M13/COS peptide expression plasmid. This plasmid design is identical topAM6 (see FIG. 1) with the exception of the following modifications:Instead of using separate signal peptide sequences for the display andsecretion modes, this design utilizes the B-lactamase signal sequence,which functions in both modes. This design eliminates the need for thefirst pair of splice signals.

[0272] An illustrative transwell cell culture chamber is shown in FIG.4A. This illustration depicts the transwell system that is utilized inthe secretion mode of the subject method. The lower compartmentcontaining the target cells is a well in, an ordinary tissue culturedish. The transwell, which fits into this well, is a chamber with solidsides and a permeable, microporous membrane bottom. The secreting COScells are grown on this membrane through which their secreted peptidescan diffuse and come into contact with the target cells.

[0273] In other embodiments, the subject method is used as part of anindicator plate antimicrobial assay. FIG. 4B illustrates a method foridentifying peptides with antimicrobial activity. Bacterial cellscarrying plasmids that direct the secretion of test peptides are platedon top of an agar embedded culture of target microorganisms. Productionof an inhibitory peptide by any of the bacterial colonies will result inan inhibition zone in the agar embedded target cell culture. This zonewill be visualized as a clear area in the agar, due to the inability ofthe target cells to form a dense culture in the presence of the secretedpeptide.

[0274] The method of the present invention has been utilized to identifyanti-angiogenic peptides. FIG. 5 is a flowchart depicting utilization ofthe M13 display/COS secretion method for identification ofanti-angiogenic peptides. FIG. 6 shows the sequence of a version of thepAM6 M13/COS peptide expression plasmid. In this example the randompeptide, flanked by distinct BstX1 sites, is actually a Mycepitope-6xHis control peptide.

[0275]FIGS. 6, 7 and 8 each provide a more detailed look at thefunctional elements of the pAM6, pAM7 and pAM8 M13/COS plasmid shown inFIGS. 1, 2 and 3 respectively. In each of these example the randompeptide, flanked by distinct BstX1 sites, is actually a Mycepitope-6xHis control peptide.

[0276] To test each of the plasmids, E. coli were transformed with thenegative control pLITMUS plasmid and the Myc epitope-6xHis encodingplasmids: pAM6, pAM7 and pAM8. The cells were grown at 37° C. to logphase and induced with 0.1 mM IPTG for 3 hours (+) or grown for 3 hoursin the absence of IPTG (−). Whole cell lysates were separated byelectrophoresis on a 16% tricine SDS-PAG, and immunoblotted withanti-myc antibody.

[0277] The results (FIG. 9) demonstrate that each of the M13/COS vectorsexpress the Myc-6xHis-pVIII fusion protein, and that the products ofpAM7 and pAM8 appear to be properly processed. However, the apparentmolecular weight of the peptides secreted from pAM6 indicates a signalpeptide processing problem, i.e., the higher molecular weight speciescorresponds to the expected size of a peptide without the signalpeptides cleaved.

[0278] The incorporation of myc-6xHis-pVIII fusion protein into phagemidcapsids was tested by anti-myc western blotting. E. coli weretransformed with the negative control pLITMUS plasmid and the Mycepitope-6xHis encoding plasmids: pAM6, pAM7 and pAM8. The cells weregrown at 37° C. to log phase, induced with 0.1 mM IPTG, infected withM13 helper phage and grown overnight. Phagemids contained in the culturemedia supernatant were separated by electrophoresis on a 16% tricineSDS-PAG, and immunoblotted with anti-myc antibody.

[0279]FIG. 10 demonstrates that the pAM7 and pAM8 vectors result inproperly processed Myc-6xHis-pVIII fusion protein being incorporatedinto the capsid. Very little fusion protein is incorporated into thecapsid of phagemids produced by pAM6 transformants, and nothing isdetected in the negative control pLITMUS lane.

[0280] The ratio of native pVIII versus myc-6xHis-pVIII proteins inphagemid capsids was also determined. As above, E. coli were transformedwith the negative control pLITMUS plasmid and the Myc epitope-6xHisencoding plasmids: pAM6, pAM7 and pAM8. The cells were grown at 37° C.to log phase, induced with 0.1 mM IPTG, infected with M13 helper phageand grown overnight. Phagemids contained in the culture mediasupernatant were separated by electrophoresis on a 16% tricine SDS-PAG,and stained with coomassie blue.

[0281] We observed that pAM7 and pAM8 vectors result in properlyprocessed Myc-6xHis-pVIII fusion protein being incorporated into thecapsid at a ratio of 1-10% of native pVIII. No fusion protein isdetected in the pAM6 or pLITMUS lanes.

[0282] Phagemids produced as described above were also tested fortitration of plaque and colony forming units generated upon phagemidrescue by serially dilution and infection into log phase E. coli. SeeFIG. 11. Infected cells were either plated on soft agar to detect plaqueforming units (p.f.u.), or on ampicillin to determine colony formingunits (c.f.u.). c.f.u. represent those phage which have packaged aplasmid DNA, whereas p.f.u. represent phage which have packaged helper aphage genome DNA.

[0283] The secretion of M13/COS plasmid encoded proteins from COS-7cells, e.g., in the secretion mode, is examined. COS-7 cells weretransfected with pIC400 negative control plasmid and with pAM7 and pAM8plasmids, which encode p27 in the random peptide insertion site. Thenormally intracellular p27 was chosen for this experiment to enableefficient western blot detection of the secreted protein in the cellmedia. 20 ul aliquots of media were collected on days 1, 2, 3 and 5following transfection, separated by SDS-PAGE and immunoblotted withanti-p27 antibody. As shown in FIG. 12, the level of secreted protein inthe media increased over the time of the experiment for both pAM7 andpAM8, although there is considerably more p27 produced by the pAM7vector design. No p27 is detected with pIC400 negative control. Purifiedp27 is included as a size marker.

[0284]FIG. 13 shows an anti-pIII western blot detection of peptidesincorporated into M13 phagemid capsids as pIII fusions. Briefly,oligonucleotides encoding the the Myc epitope-6xHis peptide, athrombospondin derived peptide (tsp: SPWSSASVTCGDGVITRIR), an α_(v)β₃integrin binding peptide containing the RGD motif (CDCRGDCFC) and thefirst kringle domain of angiostatin (K1: 80 amino acids) were insertedbetween the the BstXI sites of pAM9. In E.coli, the plasmids direct theexpression of the peptide-pIII fusion proteins. For phagemid productionthe cells were grown at 37° C. to log phase, induced with 0.1 mM IPTG,infected with M13 helper phage or an M13 helper phage that carries anamber mutation in the pIII gene and grown overnight. Phagemids containedin the culture media supernatant were separated by electrophoresis on a16% tricine SDS-PAG, and immunoblotted with anti-pill antibody. As acontrol M13K07 phage particle were used. The lower bands correspond towild type pIII proteins while the upper, higher molecular weight bandscorrespond to the peptide-pIII fusion proteins.

[0285] The data demonstrate that the pAM9 derived vectors express thepredicted peptides as pIII fusions and the fusion proteins beingincorporated into the capsid of M13 phagemids.

[0286] We observed specific binding of pAM9-K1 phagemids to bovinecapillary endothelial cells. See FIG. 14. Phagemid particles thatdisplay either the Myc epitope-6xHis peptide (pAM9-myc; negativecontrol) or the first kringle domain of angiostatin (pAM9-K1, positivecontrol) as pIII fusions were used to test the specificity of phagemidbinding to bovine capillary endothelial (BCE) cells. >90% confluent BCEcells in a well of a 6 well plate were incubated with ˜5×10¹² M13K07p.f.u./well in 2.5 ml of Peptide binding buffer (1× PBS, 1 mM CaCl₂, 10mM MgCl₂, 0.1% BSA) for 30 minutes at 37C. 10⁸ c.f.u. pAM9-myc orpAM9-K1 phagemids were added to the mix and the incubation continued for45 minutes at 37C. Excess phagemids were removed by washing the cells5×5 ml Washing buffer (2× PBS, 1 mM CaCl₂, 10 mM MgCl₂, 0.1% BSA) andphagemids bound to BCE cells were eluted by 2×1 ml 0.1N HCl pH:2.2 thatwere neutralized by the addition of 1 ml 1M Tris/Cl pH:8.0. The numberof phagemids in the elution buffer was determined by infecting TG1 cellswith aliqouts of the eluates and selecting pAM9-myc or pAM9-K1transformants on LB+Amp (M13K07 phage do not form colonies on LB+Amp).

[0287] The data demonstrate the feasibility to enrich random peptidelibraries in the display mode for peptides that specifically bind toendothelial cells.

[0288]FIG. 15 shows inhibition of BCE cell proliferation in transwellsby peptides secreted from COS-7 cells. COS-7 cells were transfected withpAM9-myc, pAM9-RGD and pAM9-K1 plasmids, respectively, that direct theexpression and secretion of the Myc epitope-6xHis, the RGD and theangiostatin first kringle domain peptides. The transfected COS-7 cellswere co-incubated in Transwells with BCE cells whose proliferation wasstimulated by 1 ng/ml bFGF. As controls, untransfected COS-7 cells andbFGF stimulated BCE cells were similarly co-incubated and syntheticMyc-6xHis and RGD peptides as well as purified KI were added to themedia at the indicated concentrations. The proliferation of the bFGFstimulated BCE cells were measured 72 hrs later using the fluorescentCyQUANT proliferation kit (Molecular Probes).

[0289] The synthetic RGD peptide and the purified KI as well as theCOS-7 secreted RGD and K1 peptides inhibited bFGF stimulated BCE cellproliferation (positive controls). The negative control Mycepitope-6xHis peptide did not have inhibitory effect on BCEproliferation. The data demonstrate the feasibility of screening randompeptide libraries iri the secretion mode for peptides that inhibit theproliferation of endothelalial cells.

[0290] All of the above-cited references and publications are herebyincorporated by reference.

[0291] Equivalents

[0292] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific method and reagents described herein. Such equivalents areconsidered to be within the scope of this invention and are covered bythe following claims.

1 8 1 527 DNA Artificial Sequence Description of Artificial SequencepAM6 M13/COS peptide expression plasmid 1 cgcaattact gtgagttagctcactcatta ggcaccccag gctttacact ttatacttcc 60 ggctcgtata ttgtgtggaattgtgagcgg ataacaattt ctagaaggaa acaggtaagt 120 atg aaa aaa tta tta ttcgca att cct tta gtt gtt cct ttc tat tct 168 Lys Lys Leu Leu Phe Ala IlePro Leu Val Val Pro Phe Tyr Ser 1 5 10 15 cac tcc gct gaa tta ctg acatcc act ttg cct ttc tct cca cag ggg 216 His Ser Ala Glu Leu Leu Thr SerThr Leu Pro Phe Ser Pro Gln Gly 20 25 30 gcc acc atg aaa tgc agc tgg gttatc ttc ttc ctg atg gca gtg gtt 264 Ala Thr Lys Cys Ser Trp Val Ile PhePhe Leu Met Ala Val Val 35 40 45 aca ggg gtc aat tca gca cca ggc gga tgggcg gcc gca gag caa aag 312 Thr Gly Val Asn Ser Ala Pro Gly Gly Trp AlaAla Ala Glu Gln Lys 50 55 60 ctc att tct gaa gag gac ttg gca cac cat caccat cac cat ctg cag 360 Leu Ile Ser Glu Glu Asp Leu Ala His His His HisHis His Leu Gln 65 70 75 cca tta tct tgg cag gta agt gct gag ggt gac gatccc ttc acc tcg 408 Pro Leu Ser Trp Gln Val Ser Ala Glu Gly Asp Asp ProPhe Thr Ser 80 85 90 aaa gca agc tgataaagtc taagcccgcc taatgagcgggctttttttt 457 Lys Ala Ser 95 tactgacatc ctcgaggcct ttctctccacaggggtagat aactgaactt gtttattgca 517 gattataatg 527 2 97 PRT ArtificialSequence Description of Artificial Sequence pAM6 2 Lys Lys Leu Leu PheAla Ile Pro Leu Val Val Pro Phe Tyr Ser His 1 5 10 15 Ser Ala Glu LeuLeu Thr Ser Thr Leu Pro Phe Ser Pro Gln Gly Ala 20 25 30 Thr Lys Cys SerTrp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly 35 40 45 Val Asn Ser AlaPro Gly Gly Trp Ala Ala Ala Glu Gln Lys Leu Ile 50 55 60 Ser Glu Glu AspLeu Ala His His His His His His Leu Gln Pro Leu 65 70 75 80 Ser Trp GlnVal Ser Ala Glu Gly Asp Asp Pro Phe Thr Ser Lys Ala 85 90 95 Ser 3 488DNA Artificial Sequence Description of Artificial Sequence pAM7 M13/COSpeptide expression plasmid 3 cgcaattact ctagagccac catg aaa tgc agc tgggtt atc ttc ttc ctg 51 Lys Cys Ser Trp Val Ile Phe Phe Leu 1 5 atg gcagtg gtt aca ggg gtc aat tca ggtaagtgag ttagctcact 98 Met Ala Val Val ThrGly Val Asn Ser 10 15 cattaggcac cccaggcttt acactttata cttccggctcgtatattgtg tggaattgtg 158 agcggataac aatttcacac aggaaacagc tatg aaa atcaaa ctg gcg tta 210 Lys Ile Lys Leu Ala Leu 20 ctc gcc ctg act tct ctttct gct ctt gca ggt cca ggc gga tgg gcg 258 Leu Ala Leu Thr Ser Leu SerAla Leu Ala Gly Pro Gly Gly Trp Ala 25 30 35 40 gcc gca gag caa aag ctcatt tct gaa gag gac ttg gca cac cat cac 306 Ala Ala Glu Gln Lys Leu IleSer Glu Glu Asp Leu Ala His His His 45 50 55 cat cac cat ctg cag cca ttatct tgg cag gta agt gct gag ggt gac 354 His His His Leu Gln Pro Leu SerTrp Gln Val Ser Ala Glu Gly Asp 60 65 70 gat ccc ttc acc tcg aaa gca agctgataaagtc taagcccgcc taatgagcgg 408 Asp Pro Phe Thr Ser Lys Ala Ser 7580 gctttttttt tactgacatc ctcgaggcct ttctctccac aggggtagat aactgaactt 468gtttattgca gattataatg 488 4 80 PRT Artificial Sequence Description ofArtificial Sequence pAM7 4 Lys Cys Ser Trp Val Ile Phe Phe Leu Met AlaVal Val Thr Gly Val 1 5 10 15 Asn Ser Lys Ile Lys Leu Ala Leu Leu AlaLeu Thr Ser Leu Ser Ala 20 25 30 Leu Ala Gly Pro Gly Gly Trp Ala Ala AlaGlu Gln Lys Leu Ile Ser 35 40 45 Glu Glu Asp Leu Ala His His His His HisHis Leu Gln Pro Leu Ser 50 55 60 Trp Gln Val Ser Ala Glu Gly Asp Asp ProPhe Thr Ser Lys Ala Ser 65 70 75 80 5 426 DNA Artificial SequenceDescription of Artificial Sequence pAM8 M13/COS peptide expressionplasmid 5 cgcaattact gtgagttagc tcactcatta ggcaccccag gctttacactttatacttcc 60 ggctcgtata ttgtgtggaa ttgtgagcgg ataacaattt ctagaaggaaagccaccatg 120 tct atc caa cac ttc cgt gtt gca tta atc cct ttc ttt gcagcg ttc 168 Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala AlaPhe 1 5 10 15 tgt tta cct gtt ttc gca ggt cca ggc gga tgg gcg gcc gcagag caa 216 Cys Leu Pro Val Phe Ala Gly Pro Gly Gly Trp Ala Ala Ala GluGln 20 25 30 aag ctc att tct gaa gag gac ttg gca cac cat cac cat cac catctg 264 Lys Leu Ile Ser Glu Glu Asp Leu Ala His His His His His His Leu35 40 45 cag cca tta tct tgg cag gta agt gct gag ggt gac gat ccc ttc acc312 Gln Pro Leu Ser Trp Gln Val Ser Ala Glu Gly Asp Asp Pro Phe Thr 5055 60 tcg aaa gca agc tgataaagtc taagcccgcc taatgagcgg gctttttttt 364Ser Lys Ala Ser 65 tactgacatc ctcgaggcct ttctctccac aggggtagataactgaactt gtttattgca 424 ga 426 6 68 PRT Artificial SequenceDescription of Artificial Sequence pAM8 6 Ser Ile Gln His Phe Arg ValAla Leu Ile Pro Phe Phe Ala Ala Phe 1 5 10 15 Cys Leu Pro Val Phe AlaGly Pro Gly Gly Trp Ala Ala Ala Glu Gln 20 25 30 Lys Leu Ile Ser Glu GluAsp Leu Ala His His His His His His Leu 35 40 45 Gln Pro Leu Ser Trp GlnVal Ser Ala Glu Gly Asp Asp Pro Phe Thr 50 55 60 Ser Lys Ala Ser 65 7 19PRT Artificial Sequence Description of Artificial SequenceThrombospondin derived peptide 7 Ser Pro Trp Ser Ser Ala Ser Val Thr CysGly Asp Gly Val Ile Thr 1 5 10 15 Arg Ile Arg 8 9 PRT ArtificialSequence Description of Artificial Sequence RGD motif 8 Cys Asp Cys ArgGly Asp Cys Phe Cys 1 5

1. A method for generating a peptide with a selected biologicalactivity, comprising the steps of: (i) providing a peptide displaylibrary comprising a variegated population of test peptides expressed onthe surface of a population of display packages; (ii) in a display mode,isolating, from the peptide display libary, a sub-population of displaypackages enriched for test peptides which have a desired bindingspecificity and/or affinity for a cell or a component thereof; (iii) ina secretion mode, simultaneously expressing the enriched test peptidesub-population under conditions wherein the test peptides are secretedand are free of the display packages; and (iv) assessing the ability ofthe secreted test peptides to regulate a biological process in a targetcell.
 2. The method of claim 1, wherein the peptide display library is aphage display library.
 3. The method of claim 2, wherein the displaypackages of the phage display library are phage particles selected froma group consisting of M13, f1, fd, If31, Ike, Xf, Pf1, Pf3, X, T4, T7,P2, P4, φX-174, MS2 and f2.
 4. The method of claim 2, wherein the phagedisplay library is generated with a filamentous bacteriophage specificfor Escherichia coli and the phage coat protein is coat protein III orcoat protein VIII.
 5. The method of claim 4, wherein the filamentousbacteriophage is selected from a group consisting of M13, fd, and f1. 6.The method of claim 1, wherein the peptide display library is abacterial cell-surface display library or a spore display library. 7.The method of claim 2, wherein test peptides are enriched from thepeptide display library in the display mode by a differential bindingmeans comprising affinity separation of test peptides which specificallybind the cell or component thereof from test peptides which do not. 8.The method of claim 7, wherein the differential binding means comprisespanning the peptide display library on whole cells.
 9. The method ofclaim 7, wherein the differential binding means comprises an affinitychromatographic means in which a component of a cell is provided as partof an insoluble matrix.
 10. The method of claim 9, wherein the insolublematrix comprises a cell surface protein attached to a polymeric support.11. The method of claim 7, wherein the differential binding meanscomprises immunoprecipitating the display packages.
 12. The method ofclaim 1, wherein the display mode enriches for test peptides which bindto a cell-type specific marker.
 13. The method of claim 1, wherein thedisplay mode enriches for test peptides which bind to a cell surfacereceptor protein.
 14. The method of claim 13, wherein the receptorprotein is a G-protein coupled receptor.
 15. The method of claim 14,wherein the G-protein coupled receptor is selected from the groupconsisting of: a chemoattractant peptide receptor, a neuropeptidereceptor, a light receptor, a neurotransmitter receptor, a cyclic AMPreceptor, and a polypeptide hormone receptor.
 16. The method of claim14, wherein the G-protein coupled receptor is selected from the groupconsisting of: α1A-adrenergic receptor, α1B-adrenergic receptor,α2-adrenergic receptor, α2B-adrenergic receptor, β1-adrenergic receptor,β2-adrenergic receptor, β3-adrenergic receptor, m1 acetylcholinereceptor (AChR), m2 AChR, m3 AChR, m4 ACHR, m5 ACHR, D1 dopaminereceptor, D2 dopamine receptor, D3 dopamine receptor, D4 dopaminereceptor, D5 dopamine receptor, A1 adenosine receptor, A2b adenosinereceptor, 5-HT1a, 5-HT1b, 5HT1-like, 5-HT1d, 5HT1d-like, 5HT1d beta,substance K (neurokinin A), fMLP receptor, fMLP-like receptor,angiotensin II type 1, endothelin ETA, endothelin ETB, thrombin, growthhormone-releasing hormone (GHRH), vasoactive intestinal peptide,oxytocin, somatostatin SSTR1 and SSTR2, SSTR3, cannabinoid, folliclestimulating hormone (FSH), leutropin (LH/HCG), thyroid stimulatinghormone (TSH), thromboxane-A2, platelet-activating factor (PAF), C5aanaphylatoxin, Interleukin 8 (IL-8) IL-8RA, IL-8RB, Delta Opioid, KappaOpioid, mip-I/RANTES, Rhodopsin, Red opsin, Green opsin, Blue opsin,metabotropic glutamate mGluR1-6, histamine H2, ATP, neuropeptide Y,amyloid protein precursor, insulin-like growth factor II, bradykinin,gonadotropin-releasing hormone, cholecystokinin, melanocyte stimulatinghormone receptor, antidiuretic hormone receptor, glucagon receptor, andadrenocorticotropic hormone II.
 17. The method of claim 13, wherein thereceptor protein is a receptor tyrosine kinase.
 18. The method of claim17, wherein the receptor tyrosine kinase is an EPH receptor.
 19. Themethod of claim 18, wherein the receptor is selected from the groupconsisting of: eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyro1,tyro4, tyro5, tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10,bsk, rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio, htk, erk andnuk receptors.
 20. The method of claim 13, wherein the receptor proteinis a cytokine receptor.
 21. The method of claim 13, wherein the receptorprotein is an MIRR receptor.
 22. The method of claim 13, wherein thereceptor protein is an orphan receptor.
 23. The method of claim 1,wherein the peptide display library includes at least 10³ different testpeptides.
 24. The method of claim 1, wherein the test peptides are 4-20amino acid residues in length.
 25. The method of claim 1, wherein eachof the test peptides are encoded by a chimeric gene comprising (i) acoding sequence for the test peptide, (ii) a coding sequence for asurface protein of the display package for displaying the test peptideson the surface of a population of display packages, and (iii) RNA splicesites flanking the coding sequence for the surface protein, wherein, inthe display mode, the chimeric gene is expressed as fusion proteinincluding the test peptide and the surface protein, whereas in thesecretion mode, the test peptide is expressed without the surfaceprotein as a result of the coding sequence for the surface protein beingremoved by RNA splicing.
 26. The method of claim 1, wherein the testpeptides are expressed by a eukaryotic cell in the secretion mode. 27.The method of claim 25, wherein the eukaryotic cell is a mammalian cell.28. The method of claim 1, wherein the target cell is a eukaryotic cell.29. The method of claim 28, wherein the eukaryotic cell is a mammaliancell.
 30. The method of claim 29, wherein the mammalian cell is a humancell.
 31. The method of claim 1, wherein the biological process includesa change in cell proliferation, cell differentiation or cell death. 32.The method of claim 1, wherein the biological process is detected bychanges in intracellular calcium mobilization.
 33. The method of claim1, wherein the biological process is detected by changes inintracellular protein phosphorylation.
 34. The method of claim 1,wherein the biological process is detected by changes in phospholipidmetabolism.
 35. The method of claim 1, wherein the biological process isdetected by changes in expression of cell-specific marker genes.
 36. Themethod of claim 13, wherein the target cell further comprises a reportergene construct containing a reporter gene in operative linkage with oneor more transcriptional regulatory elements responsive to the signaltransduction acitivity of the cell surface receptor protein, expressionof the reporter gene providing the detectable signal.
 37. The method ofclaim 36, wherein the reporter gene encodes a gene product that givesrise to a detectable signal selected from the group consisting of:color, fluorescence, luminescence, cell viability relief of a cellnutritional requirement, cell growth, and drug resistance.
 38. Themethod of claim 37, wherein the reporter gene encodes a gene productselected from the group consisting of chloramphenicol acetyltransferase, beta-galactosidase and secreted alkaline phosphatase. 39.The method of claim 37, wherein the reporter gene encodes a gene productwhich confers a growth signal.
 40. The method of claim 1, wherein thesecretion mode includes expression of the test peptides by a host cellco-cultured with the target cell.
 41. The method of claim 40, whereinthe co-cultured host and target cells are separated by a membrane whichis permeable to the test peptide.
 42. The method of claim 1, wherein thesecretion mode comprises assessing the ability of the secreted testpeptides to inhibit the biological activity of an exogenously addedcompound on the target cells.
 43. The method of claim 1, wherein: instep (ii), display packages which bind to endothelial cells areisolated; and in step (iv), the ability of the secreted test peptides toinhibit proliferation of endothelial cells is assessed.
 44. The methodof claim 43, wherein: in step (iv), the ability of the secreted testpeptides to inhibit proliferation of endothelial cells in the presenseof an angiogenic amount of an endogenous growth factor is assessed. 45.The method of claim 1, comprising the further step of converting intopeptidomimietics, one or more test peptides which regulate thebiological process in the target cell.
 46. The method of claim 1 or 45,comprising the further step of formulating, with a pharmaceuticallyacceptable carrier, one or more test peptides which regulate thebiological process in the target cell or peptidomimetics thereof.
 47. Anpeptide display library enriched for test peptides having a desiredbinding specificity and/or affinity for a cell or a component thereofand which regulate a biological process in a target cell.
 48. A vectorcomprising a chimeric gene for a chimeric protein, which chimeric genecomprises (i) a coding sequence for a test peptide, (ii) a codingsequence for a surface protein of a display package, and (iii) RNAsplice sites flanking the coding sequence for the surface protein,wherein, in a display mode, the chimeric gene is expressed as a fusionprotein including the test peptide and the surface protein such that thetest peptide can be displayed on the surface of a population of displaypackages, whereas in the secretion mode, the test peptide is expressedwithout the surface protein as a result of the coding sequence for thesurface protein being removed by RNA splicing.
 49. The vector of claim48, wherein the chimeric gene further comprises a secretion signalsequence for secretion of the test peptide in the secretion mode. 50.The vector of claim 49, wherein the secretion signal sequence causessecretion of the test peptide from eukaryotic cells.
 51. The vector ofclaim 50, wherein the eukaryotic cells are mammalian cells.
 52. Thevector of claim 48, wherein the display package is a phage.
 53. Thevector of claim 52, wherein the phage is selected from a groupconsisting of M13, f1, fd, If1, Ike, Xf, Pf1, Pf3, λ, T4, T7, P2, P4,φX-174, MS2 and f2.
 54. The vector of claim 52, wherein the phage is afilamentous bacteriophage specific for Escherichia coli and the surfaceprotein is coat protein III or coat protein VIII.
 55. The vector ofclaim 54, wherein the filamentous bacteriophage is selected from a groupconsisting of M13, fd, and f1.
 56. A vector library, each vectorcomprising a chimeric gene for a chimeric protein, which chimeric genecomprises (i) a coding sequence for a test peptide, (ii) a codingsequence for a surface protein of a display package, and (iii) RNAsplice sites flanking the coding sequence for the surface protein,wherein, in a display mode, the chimeric gene is expressed as fusionprotein including the test peptide and the surface protein such that thetest peptide can be displayed on the surface of a population of displaypackages, whereas in the secretion mode, the test peptide is expressedwithout the surface protein as a result of the coding sequence for thesurface protein being removed by RNA splicing, the vector librarycollectively encodes a variegated population of test peptides.
 57. Thevector library of claim 56, wherein the chimeric gene further comprisesa secretion signal sequence for secretion of the test peptide in thesecretion mode.
 58. The vector library of claim 57, wherein thesecretion signal sequence causes secretion of the test peptide fromeukaryotic cells.
 59. The vector library of claim 58, wherein theeukaryotic cells are mammalian cells.
 60. The vector library of claim56, wherein the display package is a phage.
 61. The vector library ofclaim 60, wherein the phage is selected from a group consisting of M13,f1, fd, If1, Ike, Xf, Pf1, Pf3, λ, T4, T7, P2, P4, φX-174, MS2 and f2.62. The vector library of claim 56, wherein the phage is a filamentousbacteriophage specific for Escherichia coli and the surface protein iscoat protein III or coat protein VIII.
 63. The vector library of claim62, wherein the filamentous bacteriophage is selected from a groupconsisting of M13, fd, and f1.
 64. The vector library of claim 56,wherein the vector library collectively encodes at least 10³ differenttest peptides.
 65. The vector library of claim 56, wherein the testpeptides are 4-20 amino acid residues in length.
 66. A cell compositioncomprising a population of cells containing the vector library of claim56.
 67. A method for generating a peptide with a selected antimicrobialactivity, comprising the steps of: (i) providing a recombinant host cellpopulation which expresses a soluble peptide library comprising avariegated population of test peptides; (ii) culturing the host cellswith a target microorganism under conditions wherein the peptide libraryis secreted and diffuses to the target microorganism; and (iii) selectedhost cells expressing test peptides that inhibit growth of the targetmicroorganism.
 68. The method of claim 67, wherein the targetmicroorganism is a bacteria.
 69. The method of claim 67, wherein thetarget microorganism is a fungus.
 70. The method of claim 67, whereinthe host cell is a bacteria.
 71. The method of claim 67, wherein thehost cells are cultured on agar embedded with the target microorganisms.72. The method of claim 67, wherein the antimicrobial activity of thetest peptide is determined by zone clearing in the agar.
 73. The methodof claim 67, wherein the peptide display library includes at least 10³different test peptides.
 74. The method of claim 67, wherein the testpeptides are 4-20 amino acid residues in length.
 75. The method of claim67, comprising the further step of converting into peptidomimietics, oneor more test peptides which inhibits growth of the target microorganism.76. The method of claim 67 or 75, comprising the further step offormulating, with a pharmaceutically acceptable carrier, one or moretest peptides or peptidomimetics which inhibit growth of the targetmicroorganism.
 77. A method for preventing or treating infection of ananimal by a microorganism, comprising administering to the animal apharmaceutical preparation of claim
 76. 78. A method for modulating anangiogenic process in an animal, comprising administering to the animala pharmaceutical preparation of claim
 46. 79. A construct as shown inFIG. 1, 2 or 3.